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Abstract:

The present invention provides a novel cancer-associated antigen that can
be used in the treatment and diagnosis of cancer. Further, the invention
provides amino acid and nucleic acid sequence of the novel antigen,
binding proteins, and immunoconjugates. The invention also relates to
diagnostic and therapeutic methods and kits.

Claims:

1-31. (canceled)

32. An isolated protein comprising a cancer-associated variant of
Mammalian Scratch, wherein the cancer-associated variant of Mammalian
Scratch is expressed on the surface of cancer cells.

33. The isolated protein according to claim 32, wherein the
cancer-associated variant of Mammalian Scratch comprises a transmembrane
domain.

34. The isolated protein according to claim 32, wherein the
cancer-associated variant of Mammalian Scratch comprises the amino acid
sequence as shown in SEQ ID NO:1 or a variant thereof.

35. The isolated protein according to claim 32, wherein the
cancer-associated variant of Mammalian Scratch comprises the amino acid
sequence as shown in SEQ ID NO:2 or a variant thereof.

36. An isolated nucleic acid sequence encoding an isolated protein
according to claim 32 or a fragment thereof.

37. The isolated nucleic acid sequence according to claim 36 having the
sequence shown in SEQ ID NO:6 or a fragment thereof.

38. The isolated nucleic acid sequence according to claim 37 having the
sequence shown in SEQ ID NO:25 or a fragment thereof.

41. A method of detecting cancer cells or monitoring cancer in a subject
having or suspected of having cancer, comprising detecting the protein
according to claim 32, or a fragment thereof, on a cell in the sample,
wherein cancer is indicated, if the protein is detected on the cell.

42. The method according to claim 41 comprising: (a) providing a sample
from the subject; (b) contacting the sample with an antibody that binds
to the protein; (c) detecting the level of the protein in the sample; and
(d) comparing the level of in the sample to a control sample, wherein
increased levels of the protein as compared to the control indicates that
the subject has cancer.

43. A method of detecting cancer cells or monitoring cancer in a subject
having or suspected of having cancer, comprising detecting the nucleic
acid sequence according to claim 36, or a fragment thereof, in the
sample, wherein cancer is indicated, if the nucleic acid sequence is
detected.

44. The method according to claim 43 comprising: (a) providing a sample
from the subject; (b) extracting the nucleic acid molecules according to
any one of claims 5 to 7 from the sample; (c) amplifying the extracted
nucleic acid molecules using the polymerase chain reaction; (d)
determining the presence of nucleic acid molecules encoding the protein;
and (e) comparing the level of the nucleic acid sequence in the sample to
a control sample, wherein increased levels of the nucleic acid sequence
as compared to the control indicates that the subject has cancer.

45. The method according to claim 44 wherein the nucleic acid molecules
are amplified using a primer having the sequence shown in SEQ ID NO:27.

46. A method of detecting cancer cells or monitoring cancer in a subject
having or suspected of having cancer comprising: (a) providing a sample
from the subject; (b) extracting nucleic acid molecules encoding wild
type scratch or the cancer-associated variant of scratch from the sample;
(c) digesting the nucleic acid molecules with a KpnI restriction enzyme;
and (d) determining the size of the digested nucleic acid molecules
wherein the presence of undigested nucleic acid molecules indicates that
the subject has a predisposition to develop cancer.

47. A pharmaceutical composition comprising an effective amount of the
isolated protein according to claim 32 or fragment thereof in admixture
with a suitable diluent or carrier.

48. The pharmaceutical composition of claim 45, further comprising an
adjuvant.

49. A pharmaceutical composition comprising an effective amount of the
isolated nucleic acid sequence of claim 36 in admixture with a suitable
diluent or carrier.

50. The pharmaceutical composition of claim 49, further comprising an
adjuvant.

51. A pharmaceutical composition comprising an effective amount of
recombinant expression vector of claim 39 in admixture with a suitable
diluent or carrier.

52. The pharmaceutical composition of claim 51, further comprising an
adjuvant.

53. A method for treating or preventing cancer in a subject comprising
preventing or decreasing the function of the cancer-associated variant of
Mammalian Scratch.

54. The method according to claim 53, wherein a binding protein that
binds to a protein according to claim 32 is used to prevent or decrease
the function of the cancer-associated variant of Mammalian Scratch.

55. The method according to claim 54, wherein the function of the
cancer-associated variant of Mammalian Scratch is prevent or decreased by
decreasing or preventing the expression of the cancer-associated variant
of Mammalian Scratch in the cell.

56. A method of identifying compounds for ability to prevent or treat
cancer, comprising the steps: (a) contacting a cell expressing the
isolated protein according to claim 32 with a test compound; (b)
determining the expression or function of the isolated protein; and (c)
comparing the expression or function of the isolated protein to a
control, wherein a decrease in expression or function of the isolated
protein as compared to the control is indicative of a compound useful to
prevent or treat cancer.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.
12/097,336 filed Nov. 12, 2008, which is a national phase entry
application of PCT/CA2006/002101 filed Dec. 21, 2006 (which designated
the U.S.) which claims the benefit of U.S. provisional application Ser.
No. 60/751,965 filed Dec. 21, 2005 (now abandoned). All of the prior
applications are incorporated herein in their entirety.

INCORPORATION OF SEQUENCE LISTING

[0002] A computer readable form of the Sequence Listing
"10241-232_SequenceListing.txt" (18,407 bytes), submitted via EFS-WEB and
created on Apr. 19, 2011, is herein incorporated by reference.

FIELD OF THE INVENTION

[0003] The invention relates to a novel antigen associated with cancer and
methods and compositions for treating and detecting cancer.

BACKGROUND OF THE INVENTION

[0004] In the year 2000, an estimated 22 million people were suffering
from cancer worldwide and 6.2 millions deaths were attributed to this
class of diseases. Every year, there are over 10 million new cases and
this estimate is expected to grow by 50% over the next 15 years (WHO,
World Cancer Report. Bernard W. Stewart and Paul Kleihues, eds. IARC
Press, Lyon, 2003). Current cancer treatments are limited to invasive
surgery, radiation therapy and chemotherapy, all of which cause either
potentially severe side-effects, non-specific toxicity and/or
traumatizing changes to ones body image and/or quality of life. Cancer
can become refractory to chemotherapy reducing further treatment options
and likelihood of success. The prognosis for some cancer is worse than
for others and some are almost always fatal. In addition, some cancers
with a relatively high treatment success rate remain major killers due to
their high incidence rates.

[0005] One of the causes for the inadequacy of current cancer treatments
is their lack of selectivity for affected tissues and cells. Surgical
resection always involves the removal of apparently normal tissue as a
"safety margin" which can increase morbidity and risk of complications.
It also always removes some of the healthy tissue that may be
interspersed with tumor cells and that could potentially maintain or
restore the function of the affected organ or tissue. Radiation and
chemotherapy will kill or damage many normal cells due to their
non-specific mode of action. This can result in serious side-effects such
as severe nausea, weight loss and reduced stamina, loss of hair etc., as
well as increasing the risk of developing secondary cancer later in life.
Treatment with greater selectivity for cancer cells would leave normal
cells unharmed thus improving outcome, side-effect profile and quality of
life.

[0006] The selectivity of cancer treatment can be improved by targeting
molecules that are specific to cancer cells and not found on normal
cells. These molecules can then be used as a target to antibody-based
diagnostic or therapeutics or for drugs capable of altering their
function.

[0007] What little is known about the wild type Scratch protein, has been
obtained on the basis of conceptual translation and analysis of the
resulting hypothetical protein sequence. Expression of Mammalian Scratch
(Scrt) mRNA has been found confined to the brain, spinal cord and newly
differentiating, postmitotic neurons suggesting a potential role in
neuronal differentiation. The human mammalian Scratch gene has been
mapped to q24.3 (chromosome 8) Nakakura et al 2001a, PNAS vol 98 p
4010-4015 and Nakakura et al 2001. Mol. Brain. Res. Vol 95 p 162-166.

[0008] Mammalian Scratch shares a SNAG domain with other zinc finger
proteins, such as SNAI1, SNAI2, SNAI3, GFII and GFIIB. While quite a few
labs working on SNAG domains (Bathe E et al. 2000. Nat. Cell Biol, Vol.
2:84-89; Kataoka H et al., 2000. Nucleic Acids Res. Vol. 28:626-633;
Grimes H L et al. 1996. Mol. Cell. Biol. Vol. 16:6263-6272; Hemavathy K
et al. 2000. Mol. Cell. Biol. Vol: 20:5087-5095) and snail locomotor
functionality have come across the over-expression of the Scrt gene, the
presence of the protein itself has not been shown thus far. Based on the
hypothetical protein sequence, the Scratch protein should have five zinc
finger domains and a SNAG domain responsible for a function in
transcription repression. The sequence indicates that the resulting
protein would be an intra-nuclear one and in fact expression of
recombinant Mammalian Scratch has been found confined to nucleus of
transfected cells (Nakakura et al 2001a, PNAS vol 98 p 4010-4015).

SUMMARY OF THE INVENTION

[0009] The present inventors have identified a novel cancer-associated
protein. Accordingly, the invention provides a novel cancer-associated
antigen that can be used in the treatment and diagnosis of cancer. In
particular, the antigen is associated with glioblastoma, melanoma, breast
cancer, lung cancer, ovarian cancer, lymphoma, colon cancer, gastric
cancers and/or prostate cancer.

[0010] The novel antigen is a variant of Mammalian Scratch. The variant
has a transmembrane domain that is absent in wild type Scratch and as a
result the protein of the invention is detectable on the cell surface.
Accordingly, the invention includes an isolated protein comprising, a
cancer-associated variant of Mammalian Scratch that is expressed on the
surface of cancer cells. In an embodiment of the invention, the
cancer-associated variant of Mammalian Scratch comprises the amino acid
sequence defined by SEQ ID NO:1 or a variant thereof, or the amino acid
sequence defined by SEQ ID NO:2 or a variant thereof.

[0011] Another aspect of the invention is an isolated protein comprising
the amino acid sequence of SEQ ID NO:1 or a variant thereof or the amino
acid sequence of SEQ ID NO:2 or a variant thereof.

[0012] The invention also includes isolated nucleic acid sequences
encoding the isolated protein of the invention, recombinant expression
vectors comprising the nucleic acid sequences of the invention and host
cells comprising the recombinant expression vectors of the invention.

[0013] In another aspect of the invention, the invention includes a method
of detecting or monitoring cancer in a subject having or suspected of
having cancer, comprising detecting the isolated protein of the invention
on a cell in the sample, wherein cancer is indicated, if the isolated
protein is detected on the cell.

[0014] In addition, the invention includes methods of detecting or
monitoring cancer in a subject having or suspected of having cancer,
comprising detecting the expression of the cancer-associated variant of
Mammalian Scratch in the cell in the sample, wherein cancer is indicated,
if the expression of the cancer-associated variant of Mammalian Scratch
is detected in the cell.

[0015] A further aspect of the invention is a method of treating or
preventing cancer in a subject by modulating the function or expression
of a Mammalian Scratch in the cancer cell.

[0016] The invention also includes pharmaceutical compositions comprising
an effective amount of the isolated proteins of the invention, the
isolated nucleic acid sequences of the invention and/or the recombinant
expression vectors of the invention.

[0017] A further aspect of the invention is the use of the isolated
proteins of the invention, the isolated nucleic acid sequences of the
invention and/or the recombinant expression vectors of the invention to
elicit an immune response in a subject.

[0018] Another aspect of the invention is the use of the isolated proteins
of the invention, the isolated nucleic acid sequences of the invention
and/or the recombinant expression vectors of the invention to treat or
prevent cancer.

[0019] In addition, the invention includes methods for treating or
preventing cancer in a subject comprising administering to the subject or
a cell from the subject an effective amount of the isolated proteins of
the invention, the isolated nucleic acid sequences of the invention
and/or the recombinant expression vectors of the invention.

[0020] The invention also includes methods for inducing an immune response
in a subject against the isolated protein of the invention comprising
administering to the subject or a cell from the subject an effective
amount of the isolated proteins of the invention, the isolated nucleic
acid sequences of the invention and/or the recombinant expression vectors
of the invention.

[0021] A further aspect of the invention is a method of detecting or
monitoring cancer in a subject, comprising the steps of: [0022] (1)
contacting a test sample taken from said subject with a binding protein
that binds specifically to an antigen on the cancer cell to produce a
binding protein-antigen complex; [0023] (2) measuring the amount of
binding protein-antigen complex in the test sample; and [0024] (3)
comparing the amount of binding protein-antigen complex in the test
sample to a control.

[0025] Other features and advantages of the present invention will become
apparent from the following detailed description. It should be
understood, however, that the detailed description and the specific
examples while indicating preferred embodiments of the invention are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will become
apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will now be described in relation to the drawings in
which:

[0027] FIG. 1 shows the glycan structures involved in the binding of
VB3-011 to the protein of the invention. Chondroitin sulphate A, also
known as Chondroitin-4-sulphate, (due to the presence of the Sulfate
molecule at position 4), is a linear molecule of repeating
D-galactosamine and glucuronic acid (A). When two such CSA molecules get
cross-linked via a 2-6 alpha linkage, the glycan unit now represents the
one recognized by Heamagglutinin (HA) (B).

[0028] FIG. 2 is a schematic representation of HA reagent immobilization.
At the first level, the specificity of HA is enhanced by blocking the
anti-HA/protein-G-sepharose epitopes. At the second level, it is
immobilized with protein-G-sepharose, simultaneously blocking any
non-specificity arising due to the anti-IgG coupling step. Level 3,
reaction with ethanolamine, ensures that apart from the HA epitope, all
other reactive amine groups are blocked, thus increasing specificity for
HA.

[0029] FIG. 3 shows the results of a lectin-based purification of the
protein of the invention that is detected by VB3-011. Con-A and WGA
lectins pulled down non-specific proteins, whereas HA pulled down only
one protein present in the positive and absent in the negative cell line
(FIG. 3A). U87MG, U118MG and A375 show a single band when purified with
HA, whereas Panc-1 and Daudi show no detectable bands (FIG. 3B).

[0030] FIG. 4 shows the disappearance of the glycan residue due to
degradation at room temperature. A 50 kDa band usually observed on IP
with HA reagent, when let to sit at room temperature for an hour prior to
separation on SDS-PAGE, resulted in the degradation of the glycan
residue, thus showing the presence of a protein band devoid of the glycan
portion of the antigen at 36 kDa.

[0031] FIG. 5 shows the presence of one single protein spot in the
purified antigen complex, at Mw-36 kDa and pI=9.7. This represents a
Western blot profile of the 2D-PAGE obtained on VB3-011 antigen
purification. The corresponding spot from the gel was used for ID
purposes.

[0032] FIG. 6 and SEQ ID NO:3 show the complete mapping of the peptides
obtained and the sequence coverage of the wild type Mammalian Scratch
molecule, Accession # gi|13775236. The underlined amino acids represent
the sequences of amino acids identified from MS analysis.

[0033] FIG. 7 and SEQ ID NO:4 show the sequence coverage obtained for
gi|15928387 from MDA-MB-435S and a BLAST sequence comparison for
435S-derived sequence and Scrt. MDA-MD-435S shows the presence of a
truncated version of Scratch, i.e., 17.823 kDa protein gi|15928387, with
100% homology to sequences 185-366 of SEQ ID NO:3 of the Wild type
Mammalian Scratch molecule.

[0034] FIG. 8 shows the TOF-MS scans of peptides obtained from A-375 cell
line, to detect the presence of all peptide ions in the sample. One
hundred scans at 1200-1400V in the range of 100-1200 amu on a static
nanospray resulted in the recovery of a significant number of peptides,
which when analyzed yielded a protein ID as Mammalian scratch. FIG. 8A
represents the TOF-MS scan with all multiply charged peptide ions and
FIG. 8B represents the deconvoluted spectrum with singly charged peptide
ions.

[0035] FIG. 9 shows TOF-MS scans of peptides obtained from U87MG cell
line, to detect the presence of all peptide ions in the sample. Three
hundred scans at 1200-1400V in the range of 100-1200 amu on a static
nanospray resulted in the recovery of a significant number of peptides,
which when analyzed yielded a protein ID as Mammalian Scratch. FIG. 9A
represents the TOF-MS scan with all multiply charged peptide ions and
FIG. 9B represents the deconvoluted spectrum with singly charged peptide
ions.

[0036] FIG. 10 shows TOF-MS scans of peptides obtained from U118MG cell
line, to detect the presence of all peptide ions in the sample.
Twenty-seven scans at 1200-1400V in the range of 100-1200 amu on a static
nanospray resulted in the recovery of a significant number of peptides,
which when analyzed yielded a protein ID as Mammalian Scratch. FIG. 10A
represents the TOF-MS scan with all multiply charged peptide ions and
FIG. 10B represents the deconvoluted spectrum with singly charged peptide
ions.

[0037] FIG. 11 and SEQ ID NO:1 show the sequence coverage of peptides
recovered from mass spectrometry analysis as listed in Table 2. A total
of 18 peptides were recovered from in-gel tryptic digestion and 67%
coverage of the protein was obtained. Underlined sequences represent the
peptide sequences recovered. The highlighted peptide includes novel
sequences. Specifically, the sequences in bold are the novel sequences
and the ones in italics represent exact matches with Mammalian Scratch.

[0038] FIG. 12 shows the peptide mass fingerprinting results for the
peptides recovered from VB3-011Ag. Protein scores greater than 77 were
considered significant. The only significant protein IDs observed pointed
to the one antigen, known as Mammalian Scratch with a score of 149.

[0039] FIG. 13 shows that the identified antigen, Mammalian Scratch, has a
significant score of 149. Due to the nature of the database server and
the similarity/homology linked proteins, all the isoforms of this protein
were pulled down as hits. MS/MS fragmentation and identity of peptides
confirms that the antigen is Mammalian Scratch.

[0041] FIG. 15 shows the MS/MS ion fragmentation of the neutral peptide
Mr. 2134.985448, appearing as a doubly charged molecule (1068.500000,
2+). The flanking regions of the recovered peptide exactly matched the
peptide from Scratch; however the rest of the sequence showed not more
than 40% homology in the sequence information. The peptide sequence is
residues 2-23 of SEQ ID NO:2.

[0043] FIG. 17 and SEQ ID NOS:5 and 3 show a restriction map of Scratch-1.

[0044] FIG. 18 shows the in vitro cytotoxicity of VB6-011 in an MTS assay
of VB6-011 with antigen-positive cells MB-435S (open circle) and
antigen-negative cells Panc-1 (black circle). Cells seeded at 1000 cells
per well, were incubated with the Fab-de-bouganin purified proteins.
After 5 days incubation, the cell viability was measured and IC50
was determined.

DETAILED DESCRIPTION OF THE INVENTION

(A) Definitions

[0045] The term "a cell" includes a single cell as well as a plurality or
population of cells. Administering an agent (such as a cancer-associated
protein) to a cell includes both in vitro and in vivo administrations.

[0046] The term "administered systemically" as used herein means that the
immunoconjugate and/or other cancer therapeutic may be administered
systemically in a convenient manner such as by injection (subcutaneous,
intravenous, intramuscular, etc.), oral administration, inhalation,
transdermal administration or topical application (such as topical cream
or ointment, etc.), suppository applications, or means of an implant. An
implant can be of a porous, non-porous, or gelatinous material, including
membranes, such as sialastic membranes, or fibers. Suppositories
generally contain active ingredients in the range of 0.5% to 10% by
weight.

[0047] The term "amino acid" includes all of the naturally occurring amino
acids as well as modified amino acids.

[0048] The term "antibody" as used herein is intended to include
monoclonal antibodies, polyclonal antibodies, and chimeric antibodies.
The antibody may be from recombinant sources and/or produced in
transgenic animals. The term "antibody fragment" as used herein is
intended to include Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers,
minibodies, diabodies, and multimers thereof and bispecific antibody
fragments. Antibodies can be fragmented using conventional techniques.
For example, F(ab')2 fragments can be generated by treating the
antibody with pepsin. The resulting F(ab')2 fragment can be treated
to reduce disulfide bridges to produce Fab' fragments. Papain digestion
can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2,
scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody
fragments and other fragments can also be synthesized by recombinant
techniques.

[0049] By "at least moderately stringent hybridization conditions" it is
meant that conditions are selected which promote selective hybridization
between two complementary nucleic acid molecules in solution.
Hybridization may occur to all or a portion of a nucleic acid sequence
molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25,
30, 40 or 50) nucleotides in length. Those skilled in the art will
recognize that the stability of a nucleic acid duplex, or hybrids, is
determined by the Tm, which in sodium containing buffers is a function of
the sodium ion concentration and temperature (Tm==81.5° C.-16.6
(Log10 [Na+])+0.41(% (G+C)-600/l), or similar equation). Accordingly, the
parameters in the wash conditions that determine hybrid stability are
sodium ion concentration and temperature. In order to identify molecules
that are similar, but not identical, to a known nucleic acid molecule a
1% mismatch may be assumed to result in about a 1° C. decrease in
Tm, for example if nucleic acid molecules are sought that have a >95%
identity, the final wash temperature will be reduced by about 5°
C. Based on these considerations those skilled in the art will be able to
readily select appropriate hybridization conditions. In preferred
embodiments, stringent hybridization conditions are selected. By way of
example the following conditions may be employed to achieve stringent
hybridization: hybridization at 5× sodium chloride/sodium citrate
(SSC)/5×Denhardt's solution/1.0% SDS at Tm -5° C. based on
the above equation, followed by a wash of 0.2×SSC/0.1% SDS at
60° C. Moderately stringent hybridization conditions include a
washing step in 3×SSC at 42° C. It is understood, however,
that equivalent stringencies may be achieved using alternative buffers,
salts and temperatures. Additional guidance regarding hybridization
conditions may be found in: Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a
Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.

[0050] The term "binding protein" as used herein refers to proteins that
specifically bind to another substance such as a cancer-associated
antigen of the invention. In an embodiment, binding proteins are
antibodies or antibody fragments.

[0051] By "biologically compatible form suitable for administration in
vivo" is meant a form of the substance to be administered in which any
toxic effects are outweighed by the therapeutic effects.

[0052] The terms "cancer-associated variant of Mammalian Scratch",
"cancer-associated antigen of the invention", "tumor-associated antigen
of the invention" or "isolated protein of the invention" as used herein
refer to a novel variant of Mammalian Scratch that is expressed on the
surface of cancer cells or a variant thereof that is also expressed on
the surface of cancer cells. In one embodiment, the novel
cancer-associated antigen has at least one transmembrane domain. In
specific embodiments, the cancer-associated antigen of Mammalian Scratch
is an isolated protein comprising the amino acid sequence defined by SEQ
ID NO:1 or an isolated protein comprising the amino acid sequence defined
by SEQ ID NO:2.

[0053] The term "cancer cell" includes cancer or tumor-forming cells,
transformed cells or a cell that is susceptible to becoming a cancer or
tumor-forming cell.

[0054] A "conservative amino acid substitution", as used herein, is one in
which one amino acid residue is replaced with another amino acid residue
without abolishing the protein's desired properties.

[0055] The term "control" as used herein refers to a sample from a subject
or a group of subjects who are either known as having cancer or not
having cancer.

[0056] The term "controlled release system" as used means the
immunoconjugate and/or other cancer therapeutic of the invention can be
administered in a controlled fashion. For example, a micropump may
deliver controlled doses directly into the area of the tumor, thereby
finely regulating the timing and concentration of the pharmaceutical
composition (see, e.g., Goodson, 1984, in Medical Applications of
Controlled Release, vol. 2, pp. 115-138).

[0057] The term "derivative of a peptide" refers to a peptide having one
or more residues chemically derivatized by reaction of a functional side
group. Such derivatized molecules include for example, those molecules in
which free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free
carboxyl groups may be derivatized to form salts, methyl and ethyl esters
or other types of esters or hydrazides. Free hydroxyl groups may be
derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen
of histidine may be derivatized to form N-im-benzylhistidine. Also
included as derivatives are those peptides which contain one or more
naturally occurring amino acid derivatives of the twenty standard amino
acids. For examples: 4-hydroxyproline may be substituted for proline;
5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be
substituted for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine.

[0058] The phrase "detecting or monitoring cancer" refers to a method or
process of determining if a subject has or does not have cancer, the
extent of cancer, the severity of cancer and/or grade of cancer.

[0059] The term "direct administration" as used herein means the cancer
therapeutic may be administered, without limitation, intratumorally,
intravascularly, and peritumorally. For example, the cancer therapeutic
may be administered by one or more direct injections into the tumor, by
continuous or discontinuous perfusion into the tumor, by introduction of
a reservoir of the cancer therapeutic, by introduction of a slow-release
apparatus into the tumor, by introduction of a slow-release formulation
into the tumor, and/or by direct application onto the tumor. By the mode
of administration "into the tumor," introduction of the cancer
therapeutic to the area of the tumor, or into a blood vessel or lymphatic
vessel that substantially directly flows into the area of the tumor, is
included.

[0060] As used herein, the phrase "effective amount" means an amount
effective, at dosages and for periods of time necessary to achieve the
desired result. Effective amounts of therapeutic may vary according to
factors such as the disease state, age, sex, weight of the animal. Dosage
regime may be adjusted to provide the optimum therapeutic response. For
example, several divided doses may be administered daily or the dose may
be proportionally reduced as indicated by the exigencies of the
therapeutic situation.

[0061] The term "eliciting an immune response" or "inducing an immune
response" as used herein means initiating, triggering, causing,
enhancing, improving or augmenting any response of the immune system, for
example, of either a humoral or cell-mediate nature. The initiation or
enhancement of an immune response can be assessed using assays known to
those skilled in the art including, but not limited to, antibody assays
(for example ELISA assays), antigen specific cytotoxicity assays and the
production of cytokines (for example ELISPOT assays). Preferably, the
isolated proteins, nucleic acid sequences or recombinant expression
vectors of the present invention, and the method of the present
invention, trigger or enhance a cellular immune response, more preferably
a T cell response.

[0062] The term "VB3-011 antibody" as used herein refers to an antibody
with the variable region of the antibody disclosed in WO 97/044461 which
has been shown to specifically bind to a variety of cancer cells and does
not significantly bind to normal tissue or cells.

[0063] The term "isolated nucleic acid sequences" as used herein refers to
a nucleic acid substantially free of cellular material or culture medium
when produced by recombinant DNA techniques, or chemical precursors, or
other chemicals when chemically synthesized. An isolated nucleic acid is
also substantially free of sequences which naturally flank the nucleic
acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid)
from which the nucleic acid is derived. The term "nucleic acid" is
intended to include DNA and RNA and can be either double stranded or
single stranded.

[0064] The term "isolated proteins" refers to a protein substantially free
of cellular material or culture medium when produced by recombinant DNA
techniques, or chemical precursors or other chemicals when chemically
synthesized. It includes the novel cancer-associated antigen of the
invention.

[0065] "Mammalian Scratch" (gi|13775236; gi|46397014; gi|13129535) is a
protein encoded by a gene that has been mapped to q24.3 of human
chromosome 8. From the analysis of the hypothetical protein sequence
based on conceptual translation, mammalian scratch has 5 zinc finger
domains and a SNAG domain. It is thought to be an intranuclear protein.
The hypothetical protein sequence is shown in SEQ ID NO:3.

[0066] The term "nucleic acid sequence" as used herein refers to a
sequence of nucleoside or nucleotide monomers consisting of naturally
occurring bases, sugars and intersugar (backbone) linkages. The term also
includes modified or substituted sequences comprising non-naturally
occurring monomers or portions thereof. The nucleic acid sequences of the
present invention may be deoxyribonucleic acid sequences (DNA) or
ribonucleic acid sequences (RNA) and may include naturally occurring
bases including adenine, guanine, cytosine, thymidine and uracil. The
sequences may also contain modified bases. Examples of such modified
bases include aza and deaza adenine, guanine, cytosine, thymidine and
uracil; and xanthine and hypoxanthine.

[0067] The term "sample" as used herein refers to any fluid, cell or
tissue sample from a subject which can be assayed for cancer.

[0068] The term "sequence identity" as used herein refers to the
percentage of sequence identity between two polypeptide sequences. In
order to determine the percentage of identity between two polypeptide
sequences, the amino acid sequences of such two sequences are aligned,
preferably using the Clustal W algorithm (Thompson, J D, Higgins D G,
Gibson T J, 1994, Nucleic Acids Res. 22 (22): 4673-4680), together with
BLOSUM 62 scoring matrix (Henikoff S, and Henikoff J. G., 1992, Proc.
Natl. Acad. Sci. USA 89: 10915-10919) and a gap opening penalty of 10 and
gap extension penalty of 0.1, so that the highest order match is obtained
between two sequences wherein at least 50% of the total length of one of
the sequences is involved in the alignment. Other methods that may be
used to align sequences are the alignment method of Needleman and Wunsch
(J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv.
Appl. Math., 1981, 2: 482) so that the highest order match is obtained
between the two sequences and the number of identical amino acids is
determined between the two sequences. Other methods to calculate the
percentage identity between two amino acid sequences are generally art
recognized and include, for example, those described by Carillo and
Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in
Computational Molecular Biology, Lesk, e.d. Oxford University Press, New
York, 1988, Biocomputing: Informatics and Genomics Projects. Generally,
computer programs will be employed for such calculations. Computer
programs that may be used in this regard include, but are not limited to,
GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN
and FASTA (Altschul et al., J. Molec. Biol., 1990: 215: 403).

[0069] The term "subject" as used herein refers to any member of the
animal kingdom, preferably a mammal, more preferably a human being. In a
preferred embodiment, the subject is suspected of having or has cancer.

[0070] As used herein, the phrase "treating or preventing cancer" refers
to inhibiting of cancer cell replication, preventing transformation of a
cell to a cancer-forming cell, inhibiting of cancer spread (metastasis),
inhibiting of tumor growth, reducing cancer cell number or tumor growth,
decreasing in the malignant grade of a cancer (e.g., increased
differentiation), or improving cancer-related symptoms.

[0071] The term "variant" as used herein includes modifications or
chemical equivalents of the amino acid and nucleotide sequences of the
present invention that perform substantially the same function as the
proteins or nucleic acid molecules of the invention in substantially the
same way. For example, variants of proteins of the invention include,
without limitation, conservative amino acid substitutions. Variants of
proteins of the invention also include additions and deletions to the
proteins of the invention. In addition, variant peptides and variant
nucleotide sequences include analogs and derivatives thereof. A variant
of the cancer-associated antigen of the invention means a protein
sequence that is expressed on cancer cells but not normal cells.

(B) Novel Cancer-Associated Antigen

[0072] The invention provides a novel cancer-associated antigen that is
expressed on the surface of cancer cells and is not significantly
expressed on the surface of normal cells. The novel cancer-associated
antigen is a variant of Mammalian Scratch. It has a transmembrane domain
that is not present in Mammalian Scratch. A sequence of the transmembrane
domain is shown in SEQ ID NO:2. A sequence of the cancer associated
variant is shown in SEQ ID NO:1.

[0073] In one embodiment, the invention provides an isolated protein
comprising the amino acid sequence defined by SEQ ID NO:1 or a variant
thereof. In another embodiment, the invention provides an isolated
protein comprising the amino acid sequence defined by SEQ ID NO:2. or a
variant thereof.

[0074] The novel cancer-associated antigen is a variant of Mammalian
Scratch that is expressed on the surface of cancer cells. Accordingly,
the invention provides an isolated protein comprising a cancer-associated
variant of Mammalian Scratch, wherein the cancer-associated variant of
Mammalian Scratch is expressed on the surface of cancer cells. In one
embodiment, the cancer-associated variant of Mammalian Scratch comprises
the amino acid sequence defined by SEQ ID NO:1. In another embodiment,
the cancer-associated variant of Mammalian Scratch comprises the amino
acid sequence defined by SEQ ID NO:2.

[0075] A person skilled in the art will appreciate that the invention
includes variants to the amino acid sequences of SEQ ID NOS:1-2 wherein
such variants are also cancer-associated antigens. Variants include
chemical equivalents to the sequences disclosed by the present invention.
Such equivalents include proteins that perform substantially the same
function as the specific proteins disclosed herein in substantially the
same way. For example, equivalents include, without limitation,
conservative amino acid substitutions.

[0076] In one embodiment, the variant amino acid sequences of the isolated
proteins of the invention have at least 50%, preferably at least 60%,
more preferably at least 70%, most preferably at least 80%, and even more
preferably at least 90% sequence identity to SEQ ID NOS:1 or 2.

[0077] The invention also provides an isolated nucleic acid sequence
encoding the isolated proteins of the invention. In one embodiment, the
isolated nucleic acid has the sequence shown in SEQ ID NO:6. In addition,
the invention includes variants to the isolated nucleic acid sequences
that encode the isolated proteins of the invention. For example, the
variants include nucleotide sequences that hybridize to the nucleic acid
sequences encoding the isolated proteins of the invention under at least
moderately stringent hybridization conditions. The variant nucleic acid
sequences will encode a protein that is a cancer-associated antigen.

[0078] The invention includes the use of the isolated proteins or
cancer-associated antigens and corresponding nucleic acid sequences For
example, the use of the isolated proteins of the invention to generate
binding proteins and immunoconjugates that can be used to treat or
prevent cancer or that can be used to detect or monitor cancer in a
subject. Accordingly, the invention includes the use of the isolated
proteins and nucleic acid sequences of the invention to treat or prevent
cancer and in the manufacture of a medicament to treat or prevent cancer
or for the diagnosis of cancer.

[0079] The invention provides a novel cancer-associated antigen that is
expressed on the surface of cancer cells and not significantly expressed
on the surface of normal cells. Thus, the novel cancer-associated antigen
can be used in therapies to treat and prevent cancer, including using the
isolated proteins of the invention to elicit an immune response in vivo.
In addition, the invention includes diagnostic methods for cancer that
comprise detecting the novel cancer-associated antigen.

[0081] One aspect of the invention is a pharmaceutical composition
comprising an effective amount of the isolated protein of the invention
in admixture with a suitable diluent or carrier. Another aspect of the
invention is a pharmaceutical a composition comprising an effective
amount of the isolated nucleic acid of the invention in admixture with a
suitable diluent or carrier. A further aspect of the invention is a
pharmaceutical composition comprising an effective amount of the
recombinant expression vector of the invention in admixture with a
suitable diluent or carrier.

[0082] For example, the pharmaceutical compositions of the invention can
be used to treat or prevent cancer. In addition, the pharmaceutical
compositions can be used to elicit an immune response in a subject
against an isolated protein of the invention.

[0083] The compositions described herein can be prepared by known methods
for the preparation of pharmaceutically acceptable compositions that can
be administered to subjects, such that an effective quantity of the
active substance is combined in a mixture with a pharmaceutically
acceptable vehicle. Suitable vehicles are described, for example, in
Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences,
20th ed., Mack Publishing Company, Easton, Pa., USA, 2000). On this
basis, the compositions include, albeit not exclusively, solutions of the
substances in association with one or more pharmaceutically acceptable
vehicles or diluents, and contained in buffered solutions with a suitable
pH and iso-osmotic with the physiological fluids.

[0084] Immunogenicity can be significantly improved if the immunizing
agents (i.e. the isolated protein of the invention, and/or nucleic acid
sequences coding therefore, and/or recombinant expression vectors) and/or
composition is, regardless of administration format, co-immunized with an
adjuvant. Commonly, adjuvants are used as a 0.05 to 1.0 percent solution
in phosphate buffered saline. Adjuvants enhance the immunogenicity of an
immunogen but are not necessarily immunogenic in of themselves. Adjuvants
may act by retaining the immunogen locally near the site of
administration to produce a depot effect facilitating a slow, sustained
release of immunogen to cells of the immune system. Adjuvants can also
attract cells of the immune system to an immunogen depot and stimulate
such cells to elicit immune response. As such, embodiments of this
invention encompass pharmaceutical compositions further comprising
adjuvants.

[0085] Adjuvants have been used for many years to improve the host immune
responses to, for example, vaccines. Intrinsic adjuvants (such as
lipopolysaccharides) normally are the components of killed or attenuated
bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which
are typically non-covalently linked to antigens and are formulated to
enhance the host immune responses. Thus, adjuvants have been identified
that enhance the immune response to antigens delivered parenterally. Some
of these adjuvants are toxic, however, and can cause undesirable
side-effects making them unsuitable for use in humans and many animals.
Indeed, only aluminum hydroxide and aluminum phosphate (collectively
commonly referred to as alum) are routinely used as adjuvants in human
and veterinary vaccines. The efficacy of alum in increasing antibody
responses to diphtheria and tetanus toxoids is well established.
Notwithstanding, it does have limitations. For example, alum is
ineffective for influenza vaccination and inconsistently elicits a cell
mediated immune response with other immunogens. The antibodies elicited
by alum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse,
which may not be optimal for protection by some vaccinal agents.

[0087] In one aspect of this invention, adjuvants useful in any of the
embodiments of the invention described herein are as follows. Adjuvants
for parenteral immunization include aluminum compounds (such as aluminum
hydroxide, aluminum phosphate, and aluminum hydroxy phosphate). The
antigen can be precipitated with, or adsorbed onto, the aluminum compound
according to standard protocols. Other adjuvants such as RIBI
(ImmunoChem, Hamilton, Mont.) can also be used in parenteral
administration.

[0088] Adjuvants for mucosal immunization include bacterial toxins (e.g.,
the cholera toxin (CT), the E. coli heat-labile toxin (LT), the
Clostridium difficile toxin A and the pertussis toxin (PT), or
combinations, subunits, toxoids, or mutants thereof). For example, a
purified preparation of native cholera toxin subunit B (CTB) can be of
use. Fragments, homologs, derivatives, and fusion to any of these toxins
are also suitable, provided that they retain adjuvant activity.
Preferably, a mutant having reduced toxicity is used. Suitable mutants
have been described (e.g., in WO 95/17211 (Arg-7-Lys CT mutant), WO
96/6627 (Arg-192-Gly LT mutant), and WO 95/34323 (Arg-9-Lys and
Glu-129-Gly PT mutant)). Additional LT mutants that can be used in the
methods and compositions of the invention include, for example
Ser-63-Lys, Ala-69-Gly, Glu-110-Asp, and Glu-112-Asp mutants. Other
adjuvants (such as a bacterial monophosphoryl lipid A (MPLA) of various
sources (e.g., E. coli, Salmonella minnesota, Salmonella typhimurium, or
Shigella flexneri, saponins, or polylactide glycolide (PLGA)
microspheres) can also be used in mucosal administration.

[0090] A subject may be immunized with a pharmaceutical composition
comprising an isolated protein of the invention, an isolated nucleic acid
sequence of the invention and/or a recombinant expression vector of the
invention by any conventional route as is known to one skilled in the
art. This may include, for example, immunization via a mucosal (e.g.,
ocular, intranasal, oral, gastric, pulmonary, intestinal, rectal,
vaginal, or urinary tract) surface, via the parenteral (e.g.,
subcutaneous, intradermal, intramuscular, intravenous, or
intraperitoneal) route or intranodally. Preferred routes depend upon the
choice of the immunogen as will be apparent to one skilled in the art.
The administration can be achieved in a single dose or repeated at
intervals.

[0091] The appropriate dosage depends on various parameters understood by
skilled artisans such as the immunogen itself (i.e. peptide vs. nucleic
acid (and more specifically type thereof)), the route of administration
and the condition of the animal to be vaccinated (weight, age and the
like).

[0092] A person skilled in the art will appreciate that the pharmaceutical
compositions can be formulated for administration to subjects in a
biologically compatible form suitable for administration in vivo. The
substances may be administered to living organisms including humans, and
animals. Administration of a therapeutically active amount of the
pharmaceutical compositions of the present invention is defined as an
amount effective, at dosages and for periods of time necessary to achieve
the desired result. For example, a therapeutically active amount of a
substance may vary according to factors such as the disease state, age,
sex, and weight of the individual, and the ability of the recombinant
protein of the invention to elicit a desired response in the individual.
Dosage regime may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered daily or
the dose may be proportionally reduced as indicated by the exigencies of
the therapeutic situation.

[0093] The pharmaceutical composition of the invention may be administered
systemically. The pharmaceutical preparation may be administered directly
to the cancer site. Depending on the route of administration, the
pharmaceutical composition may be coated in a material to protect the
composition from the action of enzymes, acids and other natural
conditions that may inactivate the compound.

[0094] In accordance with one aspect of the present invention, the
pharmaceutical composition is delivered to the subject by direct
administration. The invention contemplates the pharmaceutical composition
being administered in at least an amount sufficient to achieve the
endpoint, and if necessary, comprises a pharmaceutically acceptable
carrier.

[0095] In accordance with another aspect, the pharmaceutical composition
may be administered in vitro. For example, lymphocytes may be removed
from a subject with cancer and stimulated in vitro with the composition
and then infused back into the subject.

[0096] The invention also provides methods for reducing the risk of
post-surgical complications comprising administering an effective amount
of the pharmaceutical composition of the invention before, during, or
after surgery to treat cancer.

[0097] Pharmaceutical compositions include, without limitation,
lyophilized powders or aqueous or non-aqueous sterile injectable
solutions or suspensions, which may further contain antioxidants,
buffers, bacteriostats and solutes that render the compositions
substantially compatible with the tissues or the blood of an intended
recipient. Other components that may be present in such compositions
include water, surfactants (such as Tween), alcohols, polyols, glycerin
and vegetable oils, for example. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules, tablets, or
concentrated solutions or suspensions. The pharmaceutical compositions of
the invention may be supplied, for example but not by way of limitation,
as a lyophilized powder which is reconstituted with sterile water or
saline prior to administration to the subject.

[0098] Pharmaceutical compositions of the invention may comprise a
pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable
carriers include essentially chemically inert and nontoxic compositions
that do not interfere with the effectiveness of the biological activity
of the pharmaceutical composition. Examples of suitable pharmaceutical
carriers include, but are not limited to, water, saline solutions,
glycerol solutions, ethanol,
N-(1(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),
diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such
compositions should contain a therapeutically effective amount of the
compound, together with a suitable amount of carrier so as to provide the
form for direct administration to the subject.

[0099] The composition may be in the form of a pharmaceutically acceptable
salt which includes, without limitation, those formed with free amino
groups such as those derived from hydrochloric, phosphoric, acetic,
oxalic, tartaric acids, etc., and those formed with free carboxyl groups
such as those derived from sodium, potassium, ammonium, calcium, ferric
hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,
histidine, procaine, etc.

[0100] In various embodiments of the invention, the pharmaceutical
composition is directly administered systemically or directly to the area
of the tumor(s).

[0101] The pharmaceutical compositions may be used in methods for treating
animals, including mammals, preferably humans, with cancer. The dosage
and type of pharmaceutical composition to be administered will depend on
a variety of factors which may be readily monitored in human subjects.
Such factors include the etiology and severity (grade and stage) of the
cancer.

[0102] Clinical outcomes of cancer treatments using the pharmaceutical
compositions of the invention are readily discernable by one of skill in
the relevant art, such as a physician. For example, standard medical
tests to measure clinical markers of cancer may be strong indicators of
the treatment's efficacy. Such tests may include, without limitation,
physical examination, performance scales, disease markers, 12-lead ECG,
tumor measurements, tissue biopsy, cytoscopy, cytology, longest diameter
of tumor calculations, radiography, digital imaging of the tumor, vital
signs, weight, recordation of adverse events, assessment of infectious
episodes, assessment of concomitant medications, pain assessment, blood
or serum chemistry, urinalysis, CT scan, and pharmacokinetic analysis.
Furthermore, synergistic effects of a combination therapy comprising the
pharmaceutical compositions of the invention and another cancer
therapeutic may be determined by comparative studies with patients
undergoing monotherapy.

[0103] Another embodiment of the invention is a kit for treating or
preventing cancer comprising an effective amount of the pharmaceutical
composition of the invention, and directions for the use thereof to treat
the cancer.

[0106] Indeed, administration of an effective amount of a pharmaceutical
composition of the invention to a patient in need of such treatment may
result in reduced doses of another cancer therapeutic having clinically
significant efficacy. Such efficacy of the reduced dose of the other
cancer therapeutic may not be observed absent administration with the
pharmaceutical compositions of the invention. Accordingly, the present
invention provides methods for treating a tumor or cancer comprising
administering a reduced dose of one or more other cancer therapeutics.

[0107] Moreover, combination therapy comprising the pharmaceutical
composition of the invention to a patient in need of such treatment may
permit relatively short treatment times when compared to the duration or
number of cycles of standard treatment regimens. Accordingly, the present
invention provides methods for treating a tumor or cancer comprising
administering one or more other cancer therapeutics for relatively short
duration and/or in fewer treatment cycles.

[0108] Thus, in accordance with the present invention, combination
therapies comprising a pharmaceutical composition of the invention and
another cancer therapeutic may reduce toxicity (i.e., side effects) of
the overall cancer treatment. For example, reduced toxicity, when
compared to a monotherapy or another combination therapy, may be observed
when delivering a reduced dose of a pharmaceutical composition of the
invention and/or other cancer therapeutic, and/or when reducing the
duration of a cycle (i.e., the period of a single administration or the
period of a series of such administrations), and/or when reducing the
number of cycles.

[0109] Accordingly, the invention provides a pharmaceutical composition of
the invention further comprising one or more additional anticancer
therapeutic, optionally in a pharmaceutically acceptable carrier.

[0110] The present invention also provides a kit comprising an effective
amount of a pharmaceutical composition of the invention, optionally, in
combination with one or more other cancer therapeutic, together with
instructions for the use thereof to treat cancer.

[0111] As stated above, combination therapy with a pharmaceutical
composition of the invention may sensitize the cancer or tumor to
administration of an additional cancer therapeutic. Accordingly, the
present invention contemplates combination therapies for preventing,
treating, and/or preventing recurrence of cancer comprising administering
an effective amount of a pharmaceutical composition of the invention
prior to, subsequently, or concurrently with a reduced dose of a cancer
therapeutic. For example, initial treatment with a pharmaceutical
composition of the invention may increase the sensitivity of a cancer or
tumor to subsequent challenge with a dose of cancer therapeutic. This
dose is near, or below, the low range of standard dosages when the cancer
therapeutic is administered alone, or in the absence of a pharmaceutical
composition of the invention. When concurrently administered, the
pharmaceutical composition of the invention may be administered
separately from the cancer therapeutic, and optionally, via a different
mode of administration.

[0112] In an alternate embodiment, administration of the additional cancer
therapeutic may sensitize the cancer or tumor to pharmaceutical
composition of the invention. In such an embodiment, the additional
cancer therapeutic may be given prior to administration of a
pharmaceutical composition of the invention.

[0113] In one embodiment, the additional cancer therapeutic comprises
cisplatin, e.g., PLATINOL or PLATINOL-AQ (Bristol Myers), at a dose
ranging from approximately 5 to 10, 11 to 20, 21 to 40, or 41 to 75
mg/m2/cycle.

[0114] In another embodiment, the additional cancer therapeutic comprises
carboplatin, e.g., PARAPLATIN (Bristol Myers), at a dose ranging from
approximately 2 to 3, 4 to 8, 9 to 16, 17 to 35, or 36 to 75
mg/m2/cycle.

[0115] In another embodiment, the additional cancer therapeutic comprises
cyclophosphamide, e.g., CYTOXAN (Bristol Myers Squibb), at a dose ranging
from approximately 0.25 to 0.5, 0.6 to 0.9, 1 to 2, 3 to 5, 6 to 10, 11
to 20, or 21 to 40 mg/kg/cycle.

[0116] In another embodiment, the additional cancer therapeutic comprises
cytarabine, e.g., CYTOSAR-U (Pharmacia & Upjohn), at a dose ranging from
approximately 0.5 to 1, 2 to 4, 5 to 10, 11 to 25, 26 to 50, or 51 to 100
mg/m2/cycle. In another embodiment, the additional cancer
therapeutic comprises cytarabine liposome, e.g., DEPOCYT (Chiron Corp.),
at a dose ranging from approximately 5 to 50 mg/m2/cycle.

[0117] In another embodiment, the additional cancer therapeutic comprises
dacarbazine, e.g., DTIC or DTICDOME (Bayer Corp.), at a dose ranging from
approximately 15 to 250 mg/m2/cycle or ranging from approximately
0.2 to 2 mg/kg/cycle.

[0118] In another embodiment, the additional cancer therapeutic comprises
topotecan, e.g., HYCAMTIN (SmithKline Beecham), at a dose ranging from
approximately 0.1 to 0.2, 0.3 to 0.4, 0.5 to 0.8, or 0.9 to 1.5
mg/m2/Cycle. In another embodiment, the additional cancer
therapeutic comprises irinotecan, e.g., CAMPTOSAR (Pharmacia & Upjohn),
at a dose ranging from approximately 5 to 9, 10 to 25, or 26 to 50
mg/m2/cycle.

[0119] In another embodiment, the additional cancer therapeutic comprises
fludarabine, e.g., FLUDARA (Berlex Laboratories), at a dose ranging from
approximately 2.5 to 5, 6 to 10, 11 to 15, or 16 to 25 mg/m2/cycle.

[0120] In another embodiment, the additional cancer therapeutic comprises
cytosine arabinoside (Ara-C) at a dose ranging from approximately 200 to
2000 mg/m2/cycle, 300 to 1000 mg/m2/cycle, 400 to 800
mg/m2/cycle, or 500 to 700 mg/m2/cycle.

[0121] In another embodiment, the additional cancer therapeutic comprises
docetaxel, e.g., TAXOTERE (Rhone Poulenc Rorer) at a dose ranging from
approximately 6 to 10, 11 to 30, or 31 to 60 mg/m2/cycle.

[0122] In another embodiment, the additional cancer therapeutic comprises
paclitaxel, e.g., TAXOL (Bristol Myers Squibb), at a dose ranging from
approximately 10 to 20, 21 to 40, 41 to 70, or 71 to 135 mg/kg/cycle.

[0123] In another embodiment, the additional cancer therapeutic comprises
5-fluorouracil at a dose ranging from approximately 0.5 to 5 mg/kg/cycle,
1 to 4 mg/kg/cycle, or 2-3 mg/kg/cycle.

[0125] In another embodiment, the additional cancer therapeutic comprises
etoposide, e.g., VEPESID (Pharmacia & Upjohn), at a dose ranging from
approximately 3.5 to 7, 8 to 15, 16 to 25, or 26 to 50 mg/m2/cycle.

[0126] In another embodiment, the additional cancer therapeutic comprises
vinblastine, e.g., VELBAN (Eli Lilly), at a dose ranging from
approximately 0.3 to 0.5, 0.6 to 0.9, 1 to 2, or 3 to 3.6
mg/m2/cycle.

[0128] In another embodiment, the additional cancer therapeutic comprises
methotrexate at a dose ranging from approximately 0.2 to 0.9, 1 to 5, 6
to 10, or 11 to 20 mg/m2/cycle.

[0129] In another embodiment, a pharmaceutical composition of the
invention is administered in combination with at least one other
immunotherapeutic which includes, without limitation, rituxan, rituximab,
campath-1, gemtuzumab, and trastuzutmab.

[0131] In another embodiment, a pharmaceutical composition of the
invention is administered in combination with a regimen of radiation
therapy. The therapy may also comprise surgery and/or chemotherapy. For
example, a pharmaceutical composition of the invention may be
administered in combination with radiation therapy and cisplatin
(Platinol), fluorouracil (5-FU, Adrucil), carboplatin (Paraplatin),
and/or paclitaxel (Taxol). Treatment with a pharmaceutical composition of
the invention may allow use of lower doses of radiation and/or less
frequent radiation treatments, which may for example, reduce the
incidence of severe sore throat that impedes swallowing function
potentially resulting in undesired weight loss or dehydration.

[0135] In yet another embodiment, a pharmaceutical composition is
administered in association with a gene therapy program to treat or
prevent cancer.

[0136] Combination therapy may thus increase the sensitivity of the cancer
or tumor to the administered pharmaceutical composition of the invention
and/or additional cancer therapeutic. In this manner, shorter treatment
cycles may be possible thereby reducing toxic events. The cycle duration
may vary according to the specific cancer therapeutic in use. The
invention also contemplates continuous or discontinuous administration,
or daily doses divided into several partial administrations. An
appropriate cycle duration for a specific cancer therapeutic will be
appreciated by the skilled artisan, and the invention contemplates the
continued assessment of optimal treatment schedules for each cancer
therapeutic. Specific guidelines for the skilled artisan are known in the
art. See, e.g., Therasse et al., 2000, "New guidelines to evaluate the
response to treatment in solid tumors. European Organization for Research
and Treatment of Cancer, National Cancer Institute of the United States,
National Cancer Institute of Canada," J Natl Cancer Inst. February 2;
92(3):205-16.

[0137] It is contemplated that a pharmaceutical composition of the
invention may be administered by any suitable method such as injection,
oral administration, inhalation, transdermal or intratumorally, whereas
any other cancer therapeutic may be delivered to the patient by the same
or another mode of administration. Additionally, where multiple cancer
therapeutics are intended to be delivered to a subject, a pharmaceutical
composition of the invention and one or more of the other cancer
therapeutics may be delivered by one method, whereas other cancer
therapeutics may be delivered by another mode of administration.

[0138] The invention also provides kits comprising an effective amount of
a pharmaceutical composition of the invention, optionally, in combination
with one or more other cancer therapeutic agent, together with
instructions for the use thereof.

(ii) Diagnostic Methods

[0139] The novel cancer-associated antigen is expressed on cancer cells
and is not significantly expressed on normal cells, thus the detection of
the novel cancer-associated antigen can be used as a diagnostic method
for cancer.

[0140] One embodiment of the invention is a method of detecting or
monitoring cancer in a subject having or suspected of having cancer,
comprising detecting a cancer-associated variant of Mammalian Scratch on
a cell in the sample, wherein cancer is indicated, if the
cancer-associated variant of Mammalian Scratch is detected on the cell.

[0141] In an embodiment of the invention, a method is provided for
detecting cancer cells in a subject comprising: [0142] (a) providing a
sample from the subject; [0143] (b) detecting the level of the
cancer-associated antigen in the sample; and [0144] (c) comparing the
level of the cancer-associated antigen in the sample to a control sample,
wherein increased levels of the cancer-associated antigen as compared to
the control indicates that the subject has cancer.

[0145] The phrase "detecting the level of the cancer-associated antigen"
includes the detection of the levels of the cancer-associated antigen as
well as detection of the levels of nucleic acid molecules encoding the
cancer-associated antigen. Examples of methods for detecting proteins and
nucleic acids are discussed in greater detail below.

[0147] The term sample can be any sample containing cancer cells that one
wishes to detect including, but not limited to, biological fluids
(including blood, serum, ascites), tissue extracts, freshly harvested
cells, and lysates of cells which have been incubated in cell cultures.

[0148] The term "control sample" includes any sample that can be used to
establish a base or normal level, and may include tissue samples taken
from healthy persons or samples mimicking physiological fluid. The
control sample can also be a sample from the subject from another point
in time, e.g. prior to cancer therapy.

[0149] The method of the invention may be used in the diagnosis and
staging of the cancer. The invention may also be used to monitor the
progression of a cancer and to monitor whether a particular treatment is
effective or not. In particular, the method can be used to confirm the
absence or removal of all tumor tissue following surgery, cancer
chemotherapy, and/or radiation therapy. The methods can further be used
to monitor cancer chemotherapy and tumor reappearance.

[0150] In an embodiment, the invention contemplates a method for
monitoring the progression of cancer in a subject, comprising: [0151]
(a) providing a sample from a subject; [0152] (b) determining the level
of the cancer-associated antigen expression in the sample; [0153] (c)
repeating steps (a) and (b) at a later point in time and comparing the
result of step (b) with the result of step (c) wherein a difference in
the level of the cancer-associated antigen expression is indicative of
the progression of the cancer in the subject.

[0154] In particular, increased levels of the cancer-associated antigen at
the later time point may indicate that the cancer is progressing and that
the treatment (if applicable) is not being effective. In contrast,
decreased levels of the cancer-associated antigen at the later time point
may indicate that the cancer is regressing and that the treatment (if
applicable) is effective.

[0155] A number of techniques can be used to detect the cancer-associated
variant of Mammalian Scratch on a cell. For example, binding proteins
such as antibodies that bind to the cancer-associated variant of
Mammalian Scratch can be used in immunoassays to detect cell surface
expression of the cancer-associated variant of Mammalian Scratch. A
person skilled in the art will appreciate that a number of techniques can
be used to detect and/or quantify cell surface expression of the
cancer-associated variant of Mammalian Scratch, including, without
limitation, Western blots, immunoprecipitation followed by SDS-PAGE,
immunocytochemistry, FACS, protein arrays, and the like.

Methods for Detecting Nucleic Acid Molecules

[0156] In one embodiment, the methods of the invention involve the
detection of nucleic acid molecules encoding the cancer-associated
antigen. Those skilled in the art can construct nucleotide probes for use
in the detection of nucleic acid sequences encoding the cancer-associated
antigen in samples. Suitable probes can be prepared based on the nucleic
acid sequence shown in SEQ ID NO:6 or SEQ ID NO:25. Suitable probes
include nucleic acid molecules based on nucleic acid sequences encoding
at least 5 sequential amino acids from regions of the cancer-associated
antigen, preferably they comprise 15 to 30 nucleotides. A nucleotide
probe may be labeled with a detectable substance such as a radioactive
label which provides for an adequate signal and has sufficient half-life
such as 32P, 3H, 14C or the like. Other detectable
substances which may be used include antigens that are recognized by a
specific labeled antibody, fluorescent compounds, enzymes, antibodies
specific for a labeled antigen, and luminescent compounds. An appropriate
label may be selected having regard to the rate of hybridization and
binding of the probe to the nucleotide to be detected and the amount of
nucleotide available for hybridization. Labeled probes may be hybridized
to nucleic acids on solid supports such as nitrocellulose filters or
nylon membranes as generally described in Sambrook et al, 1989, Molecular
Cloning, A Laboratory Manual (2nd ed.). The nucleic acid probes may be
used to detect genes, preferably in human cells, that encode the
cancer-associated antigen. The nucleotide probes may also be useful in
the diagnosis of disorders involving the cancer-associated antigen, in
monitoring the progression of such disorders, or in monitoring a
therapeutic treatment. In an embodiment, the probes are used in the
diagnosis of, and in monitoring the progression of cancer, preferably
gynecological cancer.

[0157] The probe may be used in hybridization techniques to detect genes
that encode the cancer-associated antigen. The technique generally
involves contacting and incubating nucleic acids (e.g. recombinant DNA
molecules, cloned genes) obtained from a sample from a subject or other
cellular source with a probe under conditions favorable for the specific
annealing of the probes to complementary sequences in the nucleic acids.
After incubation, the non-annealed nucleic acids are removed, and the
presence of nucleic acids that have hybridized to the probe if any are
detected.

[0158] The detection of nucleic acid molecules may involve the
amplification of specific gene sequences using an amplification method
such as polymerase chain reaction (PCR), followed by the analysis of the
amplified molecules using techniques known to those skilled in the art.
Suitable primers can be routinely designed by one of skill in the art.

[0159] Hybridization and amplification techniques described herein may be
used, to assay qualitative and quantitative aspects of expression of
genes encoding the cancer-associated antigen. For example, RNA may be
isolated from a cell type or tissue known to express a gene encoding the
cancer-associated antigen, and tested utilizing the hybridization (e.g.
standard Northern analyses) or PCR techniques which are known in the art.

[0160] The primers and probes may be used in the above described methods
in situ i.e. directly on tissue sections (fixed and/or frozen) of subject
tissue obtained from biopsies or resections.

[0161] Accordingly, the present invention provides a method of detecting
cancer cells in a subject comprising: [0162] (a) providing a sample
from the subject; [0163] (b) extracting nucleic acid molecules encoding
the cancer-associated antigen or portion thereof from the sample; [0164]
(c) amplifying the extracted nucleic acid molecules using the polymerase
chain reaction; [0165] (d) determining the presence of nucleic acid
molecules encoding the cancer-associated antigen; and [0166] (e)
comparing the level of the nucleic acid molecules encoding the
cancer-associated antigen in the sample to a control sample, wherein
increased levels of the nucleic acid molecules encoding the
cancer-associated antigen as compared to the control indicates that the
subject has cancer.

[0168] The methods of the invention described herein may also be performed
using microarrays, such as oligonucleotide arrays, cDNA arrays, genomic
DNA arrays, or tissue arrays. Preferably the arrays are tissue
microarrays.

[0169] In a preferred example, an RNA expression product encoding the
cancer-associated variant of Mammalian Scratch is used to detect the
expression of the cancer-associated variant of Mammalian Scratch by the
cell. One skilled in the art will appreciate that the RNA expression
product can be detected or quantified by detecting mRNA encoding the
cancer-associated variant of Mammalian Scratch or a fragment thereof, or
oligonucleotides, cDNA, DNA, RNA, PCR products, synthetic DNA, synthetic
RNA, or other combinations of naturally occurring or modified nucleotides
which specifically and/or selectively hybridize to the mRNA encoding the
cancer-associated variant of Mammalian Scratch or a fragment thereof.

[0170] A number of methods can be used to detect and/or quantify RNA
expression of the cancer-associated variant of Mammalian Scratch by a
cell including RT-PCR, nuclease protection assays, such as ribonuclease
protection assays and S1 nuclease assays, and Northern blots and the
like.

[0171] In a particular embodiment, the inventors have prepared PCR primers
that amplify both variant and wildtype scratch (SEQ ID NO:26) or only
variant scratch (SEQ ID NO:27) as described in Example 4. Using such
primers allows one to distinguish between variant and wild type scratch.

[0172] The inventors have also determined that the sequence of wild type
Mammalian Scratch contains a KpnI restriction site at nucleotide 118 that
is not present in the cancer-associated variant. Therefore, to test if a
cancer expresses the variant, the amplified PCR product can be digested
with the KpnI restriction enzyme followed by gel electrophoresis. If the
cells being tested express wildtype Mammalian Scratch then 2 fragments of
67 bp and 93 bp will be detected. If the cells express the
cancer-associated variant then the size of the PCR product will be the
same as the undigested control.

[0173] Accordingly, the present invention provides a method of detecting
cancer cells or monitoring cancer in a subject having or suspected of
having cancer comprising: [0174] (a) providing a sample from the
subject; [0175] (b) extracting nucleic acid molecules encoding wild type
scratch or the cancer-associated variant of scratch from the sample;
[0176] (c) digesting the nucleic acid molecules with a KpnI restriction
enzyme; and [0177] (d) determining the size of the digested nucleic acid
molecules wherein the presence of undigested nucleic acid molecules
indicates that the subject has cancer.

Methods for Detecting the Cancer-Associated Antigen

[0178] In another embodiment, the methods of the invention involve the
detection of the cancer-associated antigen. In one embodiment, the
cancer-associated antigen is detected using antibodies that specifically
bind to the cancer-associated antigen. Antibodies to the
cancer-associated antigen may be prepared using techniques known in the
art.

[0179] Antibodies specifically reactive with the cancer-associated
antigen, or derivatives, such as enzyme conjugates or labeled
derivatives, may be used to detect the cancer-associated antigen in
various samples (e.g. biological materials). They may be used as
diagnostic or prognostic reagents and they may be used to detect
abnormalities in the level of protein expression, or abnormalities in the
structure, and/or temporal, tissue, cellular, or subcellular location of
the cancer-associated antigen. In vitro immunoassays may also be used to
assess or monitor the efficacy of particular therapies. The antibodies of
the invention may also be used in vitro to determine the level of
expression of a gene encoding the cancer-associated antigen in cells
genetically engineered to produce the cancer-associated antigen.

[0180] The antibodies may be used in any known immunoassays which rely on
the binding interaction between an antigenic determinant of the
cancer-associated antigen and the antibodies. Examples of such assays are
radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence,
immunoprecipitation, latex agglutination, hemagglutination, and
histochemical tests. The antibodies may be used to detect and quantify
the cancer-associated antigen in a sample in order to determine its role
in cancer and to diagnose the cancer.

[0181] In particular, the antibodies of the invention may be used in
immuno-histochemical analyses, for example, at the cellular and
subcellular level, to detect an the cancer-associated antigen, to
localize it to particular cells and tissues, and to specific subcellular
locations, and to quantitate the level of expression.

[0182] Cytochemical techniques known in the art for localizing antigens
using light and electron microscopy may be used to detect the
cancer-associated antigen. Generally, an antibody of the invention may be
labeled with a detectable substance and the cancer-associated antigen may
be localised in tissues and cells based upon the presence of the
detectable substance. Examples of detectable substances include, but are
not limited to, the following: radioisotopes (e.g., 3H, 14C,
35S, 125I, 131I), fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors), luminescent labels such as luminol;
enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase,
luciferase, alkaline phosphatase, acetylcholinesterase), biotinyl groups
(which can be detected by marked avidin e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by optical
or calorimetric methods), predetermined polypeptide epitopes recognized
by a secondary reporter (e.g., leucine zipper pair sequences, binding
sites for secondary antibodies, metal binding domains, epitope tags). In
some embodiments, labels are attached via spacer arms of various lengths
to reduce potential steric hindrance. Antibodies may also be coupled to
electron dense substances, such as ferritin or colloidal gold, which are
readily visualised by electron microscopy.

[0183] The antibody or sample may be immobilized on a carrier or solid
support which is capable of immobilizing cells, antibodies etc. For
example, the carrier or support may be nitrocellulose, or glass,
polyacrylamides, gabbros, and magnetite. The support material may have
any possible configuration including spherical (e.g. bead), cylindrical
(e.g. inside surface of a test tube or well, or the external surface of a
rod), or flat (e.g. sheet, test strip). Indirect methods may also be
employed in which the primary antigen-antibody reaction is amplified by
the introduction of a second antibody, having specificity for the
antibody reactive against the cancer-associated antigen. By way of
example, if the antibody having specificity against the cancer-associated
antigen is a rabbit IgG antibody, the second antibody may be goat
anti-rabbit gamma-globulin labeled with a detectable substance as
described herein.

[0184] Where a radioactive label is used as a detectable substance, the
cancer-associated antigen may be localized by radioautography. The
results of radioautography may be quantitated by determining the density
of particles in the radioautographs by various optical methods, or by
counting the grains.

[0185] Labeled antibodies against the cancer-associated antigen may be
used in locating tumor tissue in subjects undergoing surgery i.e. in
imaging. Typically for in vivo applications, antibodies are labeled with
radioactive labels (e.g. iodine-123, iodine-125, iodine-131, gallium-67,
technetium-99, and indium-111). Labeled antibody preparations may be
administered to a subject intravenously in an appropriate carrier at a
time several hours to four days before the tissue is imaged. During this
period unbound fractions are cleared from the subject and the only
remaining antibodies are those associated with tumor tissue. The presence
of the isotope is detected using a suitable gamma camera. The labeled
tissue can be correlated with known markers on the subject's body to
pinpoint the location of the tumor for the surgeon.

[0186] Accordingly, in another embodiment the present invention provides a
method for detecting cancer in a subject comprising: [0187] (a)
providing a sample from the subject; [0188] (b) contacting the sample
with an antibody that binds to the cancer-associated antigen; [0189] (c)
detecting the level of the cancer-associated antigen in the sample; and
[0190] (d) comparing the level of the cancer-associated antigen in the
sample to a control sample, wherein increased levels of the
cancer-associated antigen as compared to the control indicates that the
subject has cancer. (iii) Therapeutic Methods

[0191] As mentioned above, the novel cancer-associated antigen is present
on cancer cells, but not significantly on normal cells. Thus, the novel
cancer-associated antigen can be used in therapeutic methods to prevent
and treat cancer. In addition, the novel cancer-associated antigen or
fragments thereof can be used to elicit an immune response in vivo, for
example in a vaccine, or in vitro.

[0192] One embodiment of the invention is the use of an isolated protein
of the invention or fragment thereof in the manufacture of a medicament
to treat or prevent cancer. Yet another embodiment of the invention is
the use of an isolated protein of the invention or fragment thereof to
treat or prevent cancer. A further embodiment of the invention is the use
of an isolated protein of the invention or fragment thereof in the
manufacture of a medicament to elicit an immune response. Yet another
embodiment of the invention is the use of an isolated protein of the
invention or fragment thereof to elicit an immune response.

[0193] The invention also includes the use of an isolated nucleic acid
sequence of the invention in the manufacture of a medicament to treat or
prevent cancer. The invention further includes the use of an isolated
nucleic acid sequence of the invention to treat or prevent cancer. In
addition, the invention includes the use of an isolated nucleic acid
sequence of the invention in the manufacture of a medicament to elicit an
immune response. The invention further includes the use of an isolated
nucleic acid sequence of the invention to elicit an immune response.

[0194] A further embodiment of the invention is the use of the recombinant
expression vector of the invention in the manufacture of a medicament to
treat or prevent cancer. Yet another embodiment of the invention is the
use of the recombinant expression vector of the invention to treat or
prevent cancer. Also, the invention includes the use of the recombinant
expression vector of the invention in the manufacture of a medicament to
elicit an immune response in a subject. Yet another embodiment of the
invention is the use of the recombinant expression vector of the
invention to elicit an immune response in a subject.

[0195] An additional embodiment of the invention is a method of treating
or preventing cancer comprising administering an effective amount of an
isolated protein of the invention or a fragment thereof to a subject or
cell in need thereof. In addition, the invention includes a method of
treating or preventing cancer comprising administering an effective
amount of the isolated nucleic acid sequence of the invention to a
subject or cell in need thereof. Further, the invention includes a method
of treating or preventing cancer comprising administering an effective
amount of the recombinant expression vector of the invention to a subject
or cell in need thereof.

[0196] Another embodiment of the invention is a method of inducing an
immune response in a subject against an isolated protein of the
invention, comprising administering an effective amount of the isolated
protein of the invention or a fragment thereof to a subject or cell in
need thereof. In addition, the invention includes a method of inducing an
immune response in a subject against the isolated protein of the
invention, comprising administering an effective amount of the isolated
nucleic acid sequence of the invention to a subject or cell in need
thereof. Further, the invention includes a method of inducing an immune
response in a subject against the isolated protein of the invention
comprising administering an effective amount of the recombinant
expression vector of the invention to a subject or cell in need thereof.

[0197] The above methods include both in vivo and in vitro administration
of the isolated protein of the invention. For in vitro uses, the protein
can be used to stimulate lymphocytes obtained from the patient which are
then re-infused into the subject to mount an immune response against the
cancer cells expressing the cancer-associated antigen.

[0198] A further aspect of the invention is a method of treating or
preventing cancer in a subject by modulating the activity or expression
of the cancer-associated variant of Mammalian Scratch on or in a cancer
cell.

[0199] In one embodiment of the invention, the method of treating or
preventing cancer in a subject comprises preventing or decreasing the
function of the cancer-associated variant of Mammalian. In one embodiment
of the invention, a binding protein of the invention is used to prevent
or decrease the function of the cancer-associated variant of Mammalian
Scratch.

[0200] In another embodiment of the invention, the function of the
cancer-associated variant of Mammalian Scratch is prevented or decreased
by decreasing or preventing the expression of the cancer-associated
variant of Mammalian Scratch in the cell.

[0201] Standard techniques can be used to prevent or decrease the
expression of the cancer-associated variant of Mammalian Scratch in a
cell including using antisense, triple helix, or ribozyme molecules
reactive to the transcripts of the cancer-associated variant of Mammalian
Scratch gene.

[0202] For example, standard techniques can be utilized for the production
of antisense nucleic acid molecules, i.e., molecules which are
complementary to a sense nucleic acid encoding a polypeptide of interest,
e.g., complementary to the coding strand of a double-stranded cDNA
molecule or complementary to an mRNA sequence. Accordingly, an antisense
nucleic acid can hydrogen bond to a sense nucleic acid. The antisense
nucleic acid can be complementary to an entire coding strand, or to only
a portion thereof, e.g., all or part of the protein coding region (or
open reading frame). An antisense nucleic acid molecule can be antisense
to all or part of a non-coding region of the coding strand of a
nucleotide sequence encoding a polypeptide of interest. The non-coding
regions ("5' and 3' untranslated regions") are the 5' and 3' sequences
that flank the coding region and are not translated into amino acids.

[0203] An antisense oligonucleotide can be, for example, about 5, 10, 15,
20, 25, 30, 35, 40, 45 or 50 nucleotides or more in length. An antisense
nucleic acid of the invention can be constructed using chemical synthesis
and enzymatic ligation reactions using procedures known in the art. For
example, an antisense nucleic acid (e.g., an antisense oligonucleotide)
can be chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of the
duplex formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides can be
used. Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,
7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),
5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,
and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a nucleic
acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense
orientation to a target nucleic acid of interest).

[0204] Antisense nucleic acid molecules administered to a subject or
generated in situ such that they hybridize with or bind to cellular mRNA
encoding the polypeptide of interest to thereby inhibit expression, e.g.,
by inhibiting transcription and/or translation. The hybridization can be
by conventional nucleotide complementarity to form a stable duplex, or,
for example, in the case of an antisense nucleic acid molecule which
binds to DNA duplexes, through specific interactions in the major groove
of the double helix. An example of a route of administration of antisense
nucleic acid molecules of the invention includes direct injection at a
tissue site. Alternatively, antisense nucleic acid molecules can be
modified to target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be modified
such that they specifically bind to receptors or antigens expressed on a
selected cell, e.g., a T cell or brain cell, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which bind to
cell surface receptors or antigens. The antisense nucleic acid molecules
can also be delivered to cells using vectors, e.g., gene therapy vectors,
described below. To achieve sufficient intracellular concentrations of
the antisense molecules, vector constructs in which the antisense nucleic
acid molecule is placed under the control of a strong pol II or pol III
promoter are preferred.

[0206] Ribozymes are catalytic RNA molecules with ribonuclease activity
that are capable of cleaving a single-stranded nucleic acid, such as an
mRNA, to which they have a complementary region, and can also be
generated using standard techniques. Thus, ribozymes (e.g., hammerhead
ribozymes (described in Haseloff and Gerlach, 1988, Nature 334:585-591))
can be used to catalytically cleave mRNA transcripts to thereby inhibit
translation of the protein encoded by the mRNA. A ribozyme having
specificity for a nucleic acid molecule encoding a polypeptide of
interest can be designed based upon the nucleotide sequence of a cDNA
encoding a cancer-associated variant of Mammalian Scratch. For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the
nucleotide sequence of the active site is complementary to the nucleotide
sequence to be cleaved in a Cech et al. U.S. Pat. No. 4,987,071; and Cech
et al. U.S. Pat. No. 5,116,742. Alternatively, an mRNA encoding a
polypeptide of interest can be used to select a catalytic RNA having a
specific ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel and Szostak, 1993, Science 261:1411-1418.

[0207] Triple helical structures can also be generated using well known
techniques. For example, expression of a polypeptide of interest can be
inhibited by targeting nucleotide sequences complementary to the
regulatory region of the gene encoding the polypeptide (e.g., the
promoter and/or enhancer) to form triple helical structures that prevent
transcription of the gene in target cells. See generally Helene, 1991,
Anticancer Drug Des. 6(6):569-84; Helene, 1992, Ann. N.Y. Acad. Sci.
660:27-36; and Maher, 1992, Bioassays 14(12):807-15.

[0208] In various embodiments, nucleic acid compositions can be modified
at the base moiety, sugar moiety or phosphate backbone to improve, e.g.,
the stability, hybridization, or solubility of the molecule. For example,
the deoxyribose phosphate backbone of the nucleic acids can be modified
to generate peptide nucleic acids (see Hyrup et al., 1996, Bioorganic &
Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide
nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics,
in which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using standard
solid phase peptide synthesis protocols as described in Hyrup et al.,
1996, supra; Perry-O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:
14670-675.

[0209] PNAs can, for example, be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other helper
groups to PNA, by the formation of PNA-DNA chimeras, or by the use of
liposomes or other techniques of drug delivery known in the art. For
example, PNA-DNA chimeras can be generated which may combine the
advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e.g., RNAse H and DNA polymerases, to interact with
the DNA portion while the PNA portion would provide high binding affinity
and specificity. PNA-DNA chimeras can be linked using linkers of
appropriate lengths selected in terms of base stacking, number of bonds
between the nucleobases, and orientation (Hyrup, 1996, supra). The
synthesis of PNA-DNA chimeras can be performed as described in Hyrup,
1996, supra, and Finn et al., 1996, Nucleic Acids Res. 24(17):3357-63.
For example, a DNA chain can be synthesized on a support using standard
phosphoramidite coupling chemistry and modified nucleoside analogs.
Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite can be used as a link between the PNA and the 5' end of
DNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are
then coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn et al., 1996, Nucleic Acids
Res. 24(17):3357-63). Alternatively, chimeric molecules can be
synthesized with a 5' DNA segment and a 3' PNA segment (Petersen et al.,
1995, Bioorganic Med. Chem. Lett. 5:1119-1124).

[0211] Another aspect of the invention is a method to identify compounds
that are able to modulate the expression or activity of the
cancer-associated variant of Mammalian Scratch, which can be used to
prevent or treat cancer. In one embodiment of the invention, the method
for identifying a compound for ability to prevent or treat cancer
comprises the steps: [0212] (a) contacting a cell expressing a
cancer-associated variant of Mammalian Scratch with a test compound; and
[0213] (b) determining the expression or function of the
cancer-associated variant of Mammalian Scratch; and [0214] (c) comparing
the expression or function of the cancer-associated variant of Mammalian
Scratch to a control, wherein a decrease in expression or function of the
cancer-associated variant of Mammalian Scratch as compared to the control
is indicative of a compound useful to prevent or treat cancer.

(D) Binding Proteins

[0215] Another aspect of the invention is a binding protein, preferably an
antibody or antibody fragment, that binds to the isolated proteins of the
invention. Such a binding protein can be generally referred to herein as
"a binding protein of the invention", or preferably "an antibody or
antibody fragment of the invention".

[0216] In one embodiment, the invention includes a binding protein that is
specific for a cancer-associated variant of Mammalian Scratch. In a
preferred embodiment, the cancer-associated variant of Mammalian Scratch
comprises the amino acid sequence defined by SEQ ID NO:1 or a variant
thereof or the amino acid sequence defined by SEQ ID NO:2 or a variant
thereof. In another embodiment, the binding proteins bind to an isolated
protein comprising the amino acid sequence defined by SEQ ID NO:1 or a
variant thereof or the amino acid sequence defined by SEQ ID NO:2 or a
variant thereof.

[0217] In certain embodiments, the antibody or antibody fragment comprises
all or a portion of a heavy chain constant region, such as an IgG1, IgG2,
IgG3, IgG4, IgA1, IgA2, IgE, IgM or IgD constant region. Furthermore, the
antibody or antibody fragment can comprise all or a portion of a kappa
light chain constant region or a lambda light chain constant region.

[0218] The isolated proteins of the invention may be used to prepare
monoclonal or polyclonal antibodies. Conventional methods can be used to
prepare the antibodies. For example, see Goding, J. W., Monoclonal
Antibodies: Principles and Practice, 2nd Ed., Academic Press,
London, 1986.

[0220] The invention also provides compositions comprising the binding
proteins of the invention, preferably antibodies and antibody fragments,
with a pharmaceutically acceptable excipient, carrier, buffer or
stabilizer.

[0221] In addition, the binding proteins of the invention can be used in
the diagnosis of cancer.

[0222] In a preferred embodiment, the binding proteins are antibodies or
antibody fragments that bind to cancer-associated variants of Mammalian
Scratch that is expressed on the surface of cancer cells, preferably an
isolated protein comprising any one of the amino acid sequences of SEQ ID
NOS: 1 or 2. In addition, cancer cells may be evaluated to determine
their susceptibility to the treatment methods of the invention by, for
example, obtaining a sample of the cancer cells and determining the
ability of the sample to bind to the binding proteins of the invention,
preferably antibodies or antibody fragments.

[0223] Accordingly, the present invention includes diagnostic methods,
agents, and kits that can be used by themselves or prior to, during or
subsequent to the therapeutic method of the invention in order to
determine whether or not cancer cells are present that express the
antigen and can bind to the binding proteins of the invention, preferably
antibodies and antibody fragments.

[0224] In one embodiment, the invention provides a method of detecting or
monitoring cancer in a subject comprising the steps of [0225] (1)
contacting a test sample taken from said subject with a binding protein
that binds specifically to an antigen on the cancer cell to produce a
binding protein-antigen complex; [0226] (2) measuring the amount of
binding protein-antigen complex in the test sample; and [0227] (3)
comparing the amount of binding protein-antigen complex in the test
sample to a control.

[0228] In one embodiment, the antigen is a cancer-associated variant of
Mammalian Scratch, preferably an isolated protein comprising any one of
the amino acid sequences of SEQ ID NOS:1-2.

[0229] The invention further includes a kit for detecting or monitoring
cancer comprising any one of the binding proteins of the invention that
binds to an antigen on the cancer cell and instructions for the use
thereof.

[0230] For use in the diagnostic applications, the binding proteins of the
invention, preferably antibodies or antibody fragments, may be labeled
with a detectable marker such as a radio-opaque or radioisotope, such as
3H, 14C, 32P, 35S, 123I, 125I, 131I; a
fluorescent (fluorophore) or chemiluminescent (chromophore) compound,
such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme,
such as alkaline phosphatase, beta-galactosidase or horseradish
peroxidase; an imaging agent; or a metal ion. As described above, methods
of attaching a label to a binding protein, such as an antibody or
antibody fragment, are known in the art.

[0231] Another aspect of the invention is a method of detecting or
monitoring cancer in a subject comprising the steps of [0232] (1)
measuring the amount of antibodies of the invention in a test sample
taken from said subject; and [0233] (2) comparing the amount of
antibodies of the invention in the test sample to a control.

[0234] In one embodiment, the amount of antibodies of the invention is
measured by measuring the amount of antibodies of the invention in the
test sample, for example by ELISA. In another embodiment, the amount of
antibodies of the invention is measured by measuring the expression
levels of nucleic acids encoding the antibodies of the invention in the
test sample, for example by RT-PCR.

(E) Preparation of Proteins of the Invention

[0235] A person skilled in the art will appreciate that the proteins of
the invention, such as the novel cancer-associated antigen, the binding
proteins, preferably antibodies and antibody fragments, may be prepared
in any of several ways, but is most preferably prepared using recombinant
methods.

[0236] Accordingly, the nucleic acid molecules of the present invention
may be incorporated in a known manner into an appropriate expression
vector which ensures good expression of the proteins of the invention.
Possible expression vectors include but are not limited to cosmids,
plasmids, or modified viruses (e.g. replication defective retroviruses,
adenoviruses and adeno-associated viruses), so long as the vector is
compatible with the host cell used. The expression vectors are "suitable
for transformation of a host cell", which means that the expression
vectors contain a nucleic acid molecule of the invention and regulatory
sequences selected on the basis of the host cells to be used for
expression, which is operatively linked to the nucleic acid molecule.
Operatively linked is intended to mean that the nucleic acid is linked to
regulatory sequences in a manner which allows expression of the nucleic
acid.

[0237] The invention therefore contemplates a recombinant expression
vector of the invention containing a nucleic acid molecule of the
invention, or a fragment thereof, and the necessary regulatory sequences
for the transcription and translation of the inserted protein-sequence.

[0238] Suitable regulatory sequences may be derived from a variety of
sources, including bacterial, fungal, viral, mammalian, or insect genes
(for example, see the regulatory sequences described in Goeddel, Gene
Expression Technology Methods in Enzymology 185, Academic Press, San
Diego, Calif. (1990)). Selection of appropriate regulatory sequences is
dependent on the host cell chosen as discussed below, and may be readily
accomplished by one of ordinary skill in the art. Examples of such
regulatory sequences include: a transcriptional promoter and enhancer or
RNA polymerase binding sequence, a ribosomal binding sequence, including
a translation initiation signal. Additionally, depending on the host cell
chosen and the vector employed, other sequences, such as an origin of
replication, additional DNA restriction sites, enhancers, and sequences
conferring inducibility of transcription may be incorporated into the
expression vector.

[0239] The recombinant expression vectors of the invention may also
contain a selectable marker gene which facilitates the selection of host
cells transformed or transfected with a recombinant molecule of the
invention. Examples of selectable marker genes are genes encoding a
protein such as G418 and hygromycin which confer resistance to certain
drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly
luciferase, or an immunoglobulin or portion thereof such as the Fc
portion of an immunoglobulin preferably IgG. Transcription of the
selectable marker gene is monitored by changes in the concentration of
the selectable marker protein such as β-galactosidase,
chloramphenicol acetyltransferase, or firefly luciferase. If the
selectable marker gene encodes a protein conferring antibiotic resistance
such as neomycin resistance transformant cells can be selected with G418.
Cells that have incorporated the selectable marker gene will survive,
while the other cells die. This makes it possible to visualize and assay
for expression of recombinant expression vectors of the invention and in
particular to determine the effect of a mutation on expression and
phenotype. It will be appreciated that selectable markers can be
introduced on a separate vector from the nucleic acid of interest.

[0240] The recombinant expression vectors may also contain genes which
encode a fusion moiety which provides increased expression of the
recombinant protein; increased solubility of the recombinant protein; and
aid in the purification of the target recombinant protein by acting as a
ligand in affinity purification. For example, a proteolytic cleavage site
may be added to the target recombinant protein to allow separation of the
recombinant protein from the fusion moiety subsequent to purification of
the fusion protein. Typical fusion expression vectors include pGEX (Amrad
Corp., Melbourne, Australia), pMal (New England Biolabs, Beverly, Mass.)
and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione
S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the recombinant protein.

[0241] Recombinant expression vectors can be introduced into host cells to
produce a transformed host cell. The terms "transformed with",
"transfected with", "transformation" and "transfection" are intended to
encompass introduction of nucleic acid (e.g. a vector) into a cell by one
of many possible techniques known in the art. The term "transformed host
cell" as used herein is intended to also include cells capable of
glycosylation that have been transformed with a recombinant expression
vector of the invention. Prokaryotic cells can be transformed with
nucleic acid by, for example, electroporation or calcium-chloride
mediated transformation. For example, nucleic acid can be introduced into
mammalian cells via conventional techniques such as calcium phosphate or
calcium chloride co-precipitation, DEAE-dextran mediated transfection,
lipofectin, electroporation or microinjection. Suitable methods for
transforming and transfecting host cells can be found in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor
Laboratory Press, 2001), and other laboratory textbooks.

[0242] Suitable host cells include a wide variety of eukaryotic host cells
and prokaryotic cells. For example, the proteins of the invention may be
expressed in yeast cells or mammalian cells. Other suitable host cells
can be found in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990). In addition,
the proteins of the invention may be expressed in prokaryotic cells, such
as Escherichia coli (Zhang et al., Science 303(5656): 371-3 (2004)). In
addition, a Pseudomonas based expression system such as Pseudomonas
fluorescens can be used (US Patent Application Publication No. US
2005/0186666, Schneider, Jane C et al).

[0243] Yeast and fungi host cells suitable for carrying out the present
invention include, but are not limited to Saccharomyces cerevisiae, the
genera Pichia or Kluyveromyces and various species of the genus
Aspergillus. Examples of vectors for expression in yeast S. cerevisiae
include pYepSec1 (Baldari. et al., Embo J. 6:229-234 (1987)), pMFa
(Kurjan and Herskowitz, Cell 30:933-943 (1982)), pJRY88 (Schultz et al.,
Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, San Diego,
Calif.). Protocols for the transformation of yeast and fungi are well
known to those of ordinary skill in the art (see Hinnen et al., Proc.
Natl. Acad. Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology 153:163
(1983), and Cullen et al. (Bio/Technology 5:369 (1987)).

[0245] Given the teachings provided herein, promoters, terminators, and
methods for introducing expression vectors of an appropriate type into
plant, avian, and insect cells may also be readily accomplished. For
example, within one embodiment, the proteins of the invention may be
expressed from plant cells (see Sinkar et al., J. Biosci (Bangalore)
11:47-58 (1987), which reviews the use of Agrobacterium rhizogenes
vectors; see also Zambryski et al., Genetic Engineering, Principles and
Methods, Hollaender and Setlow (eds.), Vol. VI, pp. 253-278, Plenum
Press, New York (1984), which describes the use of expression vectors for
plant cells, including, among others, PAPS2022, PAPS2023, and PAPS2034).

[0246] Insect cells suitable for carrying out the present invention
include cells and cell lines from Bombyx, Trichoplusia or Spodotera
species. Baculovirus vectors available for expression of proteins in
cultured insect cells (SF 9 cells) include the pAc series (Smith et al.,
Mol. Cell. Biol. 3:2156-2165 (1983)) and the pVL series (Luckow, V. A.,
and Summers, M. D., Virology 170:31-39 (1989)). Some baculovirus-insect
cell expression systems suitable for expression of the recombinant
proteins of the invention are described in PCT/US/02442.

[0248] The proteins of the invention may also be prepared by chemical
synthesis using techniques well known in the chemistry of proteins such
as solid phase synthesis (Merrifield, J. Am. Chem. Assoc. 85:2149-2154
(1964); Frische et al., J. Pept. Sci. 2(4): 212-22 (1996)) or synthesis
in homogenous solution (Houbenweyl, Methods of Organic Chemistry, ed. E.
Wansch, Vol. 15 I and II, Thieme, Stuttgart (1987)).

[0249] N-terminal or C-terminal fusion proteins comprising the proteins of
the invention conjugated with other molecules, such as proteins may be
prepared by fusing, through recombinant techniques. The resultant fusion
proteins contain a protein of the invention fused to the selected protein
or marker protein as described herein. The recombinant protein of the
invention may also be conjugated to other proteins by known techniques.
For example, the proteins may be coupled using heterobifunctional
thiol-containing linkers as described in WO 90/10457,
N-succinimidyl-3-(2-pyridyldithio-proprionate) or N-succinimidyl-5
thioacetate. Examples of proteins which may be used to prepare fusion
proteins or conjugates include cell binding proteins such as
immunoglobulins, hormones, growth factors, lectins, insulin, low density
lipoprotein, glucagon, endorphins, transferrin, bombesin,
asialoglycoprotein glutathione-S-transferase (GST), hemagglutinin (HA),
and truncated myc.

[0250] Accordingly, the invention provides a recombinant expression vector
comprising the nucleic acid sequences that encode the proteins of the
invention, such as the isolated proteins of the invention. Further, the
invention provides a host cell comprising the recombinant expression
vector of the invention.

[0251] The following non-limiting examples are illustrative of the present
invention:

EXAMPLES

Example 1

Isolation and Identification of Cancer Associated Scratch

Experimental Design

[0252] Melanoma cell line (A-375), glioma cell lines (U118MG and U87MG),
breast cancer cell line (MDA-MB 435S), pancreatic cell line, (PANC-1) and
T-cell line (Daudi) were used in the study (Table 1). These cell lines
were selected based on the results of tumor cell line profiling by flow
cytometry.

Growth and Maintenance of Tumor Cell Lines

[0253] The cell lines in the study were purchased from ATCC and cultured
in accordance with the guidelines and recommendations of ATCC. Cells were
harvested at 90% confluence with viability >90%.

Preliminary Characterization of the Antigen Binding to VB3-011

[0254] Preliminary characterization data was obtained from experiments
designed to assess the feasibility of the gel-based approach by dot blot
assays; and from experiments performed to determine the nature of the
epitope associated with the antigens.

[0255] The data from these experiments classified the VB3-011 antigen as a
"non-blottable" antigen with a glycan modification, i.e., the epitope
involved in binding to VB3-011 on the antigen was glycosylated.

VB3-011 Ag Enrichment and Purification

[0256] The preliminary data from the blottability study specified a
lectin-based purification method as the best antigen preparation method
for VB3-011. Extensive experimentation revealed that the glycan
modification involved a soluble form of CS (chondroitin sulphate); two of
these (CSB and CSE) have limited tissue distribution. As such, the glycan
modification could be attributable to CSA and to a lesser extent
hyaluronic acid.

[0257] Chondroitin sulphate A (CSA) is made up of linear repeating units
containing D-galactosamine and D-glucuronic acid. The amino group of
galactosamines in the basic unit of chondroitin sulfate A is acetylated,
yielding N-acetyl-galactosamine; there is a sulfate group esterified to
the 4-position in N-acetyl-galactosamine (FIG. 1A) (Sugahara K et al.
1988. J. Biol. Chem. Vol. 263:10168-10174; Sugahara K et al. 1991. Eur.
J. Biochem. Vol. 202:805-811; Prydz K and Dalen K T. 2000. J. Cell Sci.
Vol. 113:193-205). When these linear repeating units get cross-linked
(α 2-6) at branch points at C2 of the second and C6 of the first
carbon chains, such that a single unit of glycan representing more than
one linear chains of CSA are present, except for the sulfation, it
resembles the glycan, Neu5Ac (α 2→6) Gal(β 1→4)
Glucuronate, recognized by HA (FIG. 1B).

[0258] Two or more CSA molecules when cross-linked together resemble the
glycan--Neu5Ac (α 2→6) Gal (β 1→4) Glucuronate,
recognized by Hemagglutinin (HA), Azumi et al., (1991) showed that the
activity of a hemagglutinin isolated from hemocytes of the ascidian,
Halocynthia roretzi was inhibited by heparin, chondroitin sulfate, and
lipopolysaccharide (LPS), but not by mono- and disaccharides such as
N-acetyl-galactosamine, galactose, and melibiose. The hemagglutinin
showed binding ability to heparin, chondroitin sulfate and LPS, as
demonstrated by heparin-Sepharose chromatography and centrifugation
experiments, respectively (Ajit Varki et al eds. 1999. Essentials of
Glycobiology). Similarly, a Hemagglutinin from mycobacterium was shown to
bind to heparan sulfate and Hemagglutinin from Hemophilus influenzae
binds to CSA with an additional α 2-6 linkage (Azumi K et al.
A1991. Dev. Comp. Immunol. Vol. 15(1-2):9-16; Menozzi F D et al. 11996.
J. Exp. Med., Vol. 184(3):993-1001). Heparan sulfate and Chondroitin
sulfate A differ in C5 epimerization. Therefore, a new reagent that would
enable lectin-based purification was generated as follows. Recombinant HA
was immobilized to anti-HA antibody by coupling with Dimethylpimelimidate
(DMP), such that when used as an IP agent, HA recognizes the CSA
associated with the antigen on the cell surface. Membrane preparations
were affinity purified with immobilized-HA, and the eluates subjected to
SDS-PAGE and WB analysis, subsequently probed with VB3-011 antibody.

Lectin-Based Purification

[0259] Recombinant HA molecule that binds specifically to the
glycan--Neu5Ac (α 2→6) Gal (β 1→4) Glc, was made
to bind to anti-HA antibody for 2 hours at room temperature on the
nutator, followed by binding of the HA-anti-HA complex to
Protein-G-sepharose. This was followed by a centrifugation step to get
rid of the unbound fraction. The immobilized complex was then
cross-linked using Dimethylpimelimidate (DMP) that is known cross-link
proteins present in close proximities. The excess or unused cross-linker
and the unbound material were removed by a brief centrifugation step. The
non-specific amine groups that could have arisen as a by-product of the
cross-linking step were neutralized with Triethanolamine for two hours at
room temperature. The lectin-based reagent thus created was washed
thoroughly with PBS and stored with PBS containing 0.05% NaN3 at
2-8° C. Apart from the HA-reagent, Con-A-agarose and WGA-agarose
were also used as affinity purification reagents to detect better antigen
recovery.

[0260] A minimum of 500 μg membrane protein was used for the
lectin-based purification. A pre-clearing step using protein-G sepharose
alone was the first step in the purification of the antigen prior to the
addition of the reagent. A total of 15-20 μL of the reagent was used
as the precipitating agent in the mixture. The antigen-lectin mixtures
were nutated overnight at 4° C. using buffer conditions that
mimicked physiologic conditions. Care was taken to ensure that protease
inhibitors were used in every step of the antigen isolation process.

[0261] Antigen-lectin complexes were centrifuged, washed with RIP-A lysis
buffer and eluted with 0.2 M glycine pH 2.5. Supernatants representing
the unbound fractions were stored to test the proteins that were not
isolated by affinity purification. Lectin-based purifications were
carried out on two glioma cell lines (U118MG and U87MG), one melanoma
cell line (A-375), one epithelial cell line (MDA-MB-435S) and two
negative cell lines (Panc-1; and Daudi).

Gel-Based Analysis and Western Blotting

[0262] 1D-PAGE: The purified proteins were subjected to reducing
conditions of sample preparation and were subsequently analyzed by
SDS-PAGE/Western Blotting. When reducing conditions were used, the
isolated antigens were treated with sample buffer containing 1%
β-mercaptoethanol at 65° C. for 15 minutes. The resulting
blots were probed with VB3-011 and corresponding secondary antibodies
conjugated to HRP, to visualize the purified proteins by
chemiluminescence.

[0263] 2D-PAGE: The purified proteins were separated by two-dimensional
gel electrophoresis to resolve any protein stacking effect that may have
occurred in the 1D-PAGE analysis. The 2D-gel electrophoresis resolved
proteins according to their isoelectric points (pI) in the first
dimension and on the basis of their molecular weights in the second
dimension. Proteins thus resolved were transferred to nitrocellulose
membranes, overnight, and processed as in the case of 1D-PAGE. Western
blots were probed with VB3-011 and reacting proteins visualized by
chemiluminescence.

Peptide Extraction and Antigen ID

[0264] Peptide extraction from in-gel and in-solution tryptic digests:
Tryptic digestions were performed with sequencing grade trypsin in a
20-hour peptide extraction process finally resulting in the extraction of
peptides that were analyzed on a QSTAR Pulsar-I (ESI-qTOF-MS/MS),
equipped with a nanosource with a working flow rate of 20-50 nL/min. The
peptides ionize and are detected as doubly, triply or quadruply charged
molecules which are then refined to their respective masses. De-novo
sequencing of the identified proteins was also performed whenever
possible. Peptides were extracted from both positive and negative cell
lines to ensure it was the right antigen. Peptide masses extracted from
the mass spectra were used directly to identify the antigen according to
the MOWSE scores obtained on protein databases that are accessible
through the MASCOT search engine. Peptides were extracted both from gel
slices and in-solution (U118MG, U87MG, A-375, 435S) and subjected them to
MS analysis.

Results

HA Reagent Immobilization

[0265] Recombinant HA molecule is not an antibody and therefore does not
bind to protein-G-sepharose directly as an immobilizing partner. In order
to make it possible for this molecule to be functional in antigen
purification processes, HA was bound to anti-HA antibody that would bind
specifically to HA, the molecule was immobilized using
protein-G-sepharose in a sequential manner. This would not only
immobilize the complex but would block any non-specific interaction that
could arise from the presence of the anti-HA, as shown schematically in
FIG. 2. The immobilized HA-anti-HA complex was thereafter stabilized
using Dimethyl pimelimidate, a cross-linking agent that maintained the
proximities of the various reactants. The final complex generated a few
reactive amines in the process, other than the reactive binding site on
the HA molecule. These reactive groups were blocked permanently using 1M
triethanolamine, thus ensuring the maximal exposure of the reactive site
on the HA molecule.

Lectin-Purification

[0266] All purification reactions were performed with pre-cleared
proteins. Longer incubation times were used to minimize non-specificity
and enhance the stability of cognate antigen-antibody complexes. Six cell
lines (A-375, U118MG, U87MG, MDA-MB-435S, Panc-1 and Daudi) were used in
the study. Reducing conditions for sample preparations were employed
prior to the resolution of the antigens isolated on SDS-PAGE. The Western
blots were probed with VB3-011 to ensure that the antigen purified is the
cognate binding partner for VB3-011.

1D-PAGE/Western Analysis

[0267] When HA reagent was used, only one specific band was detected after
separation on a 1D-PAGE at ˜50 kDa under reducing conditions (FIG.
3A) in antigen-positive cell line (A-375), that was absent in the
negative cell line (Panc-1). Non-specific interactions were observed with
Con-A and WGA lectins indicating that the glycan present on the VB3-011
antigen was the one recognized by HA. Glioma cell line (U118MG and U87MG)
also showed the presence of a single band at ˜50 kDa when purified
using the HA reagent (FIG. 3B). When samples were allowed to sit at room
temperature for 1 hour prior to their separation on SDS-PAGE, a
predominant band at ˜36 kDa and a faint 50 kDa band were observed
in antigen-positive cell line (A-375, U118MG and U87MG) (FIG. 4).

2D-PAGE Analysis

[0268] In order to determine isoelectric points (pI) and assess the
possibility of protein stacking in the 1D-PAGE analysis, the antigens
purified by HA were separated on two-dimensional polyacrylamide gel
electrophoresis (2D-PAGE), where the separation in the first dimension is
on the basis of pI and the second dimension on the basis of molecular
weight. The gels were then transferred to nitrocellulose membranes and
subjected to standard Western blotting processing. Since the amounts
required for the detection of proteins on a 2D gel is ˜4 times
higher than the requirement for a 1D gel, purified antigens from 4
separate reactions were pooled together for one 2D-PAGE analysis. Two
separate gels were processed simultaneously for Western blot analysis to
ensure that the proteins detected on the Coomassie stained gels are the
same as those observed in the Western blots. The 2D Western blots were
probed with VB3-011 and detected by ECL (chemiluniescence). As can be
seen in FIG. 5, one single spot was detected at ˜36
kDa/pI=9.7±0.2.

Peptide Extraction and Protein Analysis

[0269] A-375, U 87MG and U118 MG membranes were used to purify antigen(s)
that bind specifically to VB3-011. A ˜50 kDa band was observed in
all three cell lines as shown in FIGS. 3A and 3B. The protein bands were
excised from the coomassie stained gels and used in-gel digestion to
extract peptides for MS analysis.

[0270] Proteins from 1D-gel band and 2D-spots were digested with trypsin
to release them from the gel and analyzed on a reverse-phase LC-MS/MS
system. The identities of the proteins were revealed by database analysis
using bioinformatic tools. Raw data included peptides obtained as listed
in the TOF-MS spectra, MS/MS fragmentation data, and a list of suggested
proteins including contaminants that do not match the pI or the molecular
weight of the protein isolated. To obtain the analysis MS/MS spectra were
submitted directly to Mascot search engines available at
www.Matrixscience.com.

Mass Spectral Analysis

[0271] Peptide analysis was done in two ways: [0272] All the peptides
recovered and reconstructed to their right masses were used directly in a
peptide mass fingerprinting step to obtain an ID for the protein. [0273]
Peptides that were abundant and well ionized were chosen for further
MS/MS ion fragmentation, wherein, the `y` and `b` ions were used to
deduce their primary structure. These sequences were then searched for
homologies in the protein database for protein ID.

[0274] Peptides ionize and are detected as doubly, triply or quadruply
charged molecules, on a LC-MS/MS system as opposed to detection as singly
charged on Matrix assisted ionization such as in MALDI. Differentially
charged peptides were thereafter refined to their respective masses, in
the mass reconstruction step. These peptide masses were then directly
analyzed by a matrix science based mascot search engine for antigen ID.
Peptide masses extracted from the mass spectra were used directly to
identify the antigen according to the MOWSE scores obtained on protein
databases that are accessible through search engines such as MASCOT,
SEQUEST, and Prospector. QSTAR-pulsar-I was used and selected for all
protein identities, because it includes the most recent protein database
additions from Pepsea is compatible with MASCOT.

Analysis of 2D Spot

[0275] Protein spot excised from the 2D-gel identified Scratch. The pI and
the molecular weight clearly matched Mammalian Scratch. A total of 37%
sequence coverage with 15 matching peptides, each peptide showing 100%
homology to the original protein was recovered (See FIG. 6).

Analysis of the 50 kDa Band Purified from the Glioma and Melanoma Cell
Lines

[0276] The data obtained from the mass spectra of all three cell lines,
(U87MG, U118MG and A375) point towards Mammalian Scratch as the antigen
that binds to VB3-011. Of all the cell lines screened, glioma cell lines
(U87MG and U118MG) showed the highest scoring identities. A-375, a
melanoma cell line also showed an over-expression of the antigen. Apart
from the above mentioned cell lines, epithelial cell lines such as
MDA-MB-435S, PC-3, A-549 and CFPAC-1 were also screened in the same
manner, but except for MDA-MB-435S, which showed the presence of a
truncated version of Scratch, i.e., 17.823 kDa protein gi|15928387, with
100% homology to sequences 158-366 of the original scratch molecule. See
FIG. 7 (SEQ ID NO:4). The membrane preparations from each of these cell
lines were used to affinity purify the VB3-011 antigen using the
HA-reagent. Rest of the epithelial cell lines showed no detectable
proteins.

[0277] TOF-MS scans were obtained both on a manual mode and an IDA mode to
recover the maximum number of peptides for a significant ID. See FIGS.
8-10.

[0278] The list of peptides recovered and their mapped positions to the
sequence from Mammalian Scratch are as given in FIG. 11 (SEQ ID NO:1) and
Table 2 (SEQ ID NOS:2 and 7 to 24). All peptides represented were
obtained by de novo sequencing.

MS/MS Fragmentation of Peptide 2402.1206 and 2134.9614

[0279] A discrete nanospray head installed on a nanosource was used for
the purpose. The collision energy was 48V, curtain gas and CAD gas were
maintained at 25 and 6, respectively, and the sample allowed to cycle for
1.667 minutes (100 cycles) to obtain stable mass ion fragmentation. MS/MS
fragmentation of two of the peptides (2402.978172-802.00000, 3+;
2134.985448-1068.500000, 2+) gave rise to the fragment ions shown in
FIGS. 14 and 15. While one of the peptides, `PELATAAGGYINGDAAVSEGYAADAF`
(SEQ ID NO:7) from peptide mass 2402.97812, mapped 100% to a sequence
from Scratch, peptide, RFLAAFLAAAGPFGFALGPSSV (SEQ ID NO:2), from peptide
mass 2134.985448, showed 100% homology in the flanking sequences but not
with the sequence in the middle, indicating an identification of a novel
sequence. The presence of this sequence is responsible for the only
transmembrane domain available on the protein. Mammalian Scratch sequence
available in the database is a result of conceptual translation and does
not have any transmembrane domains in the sequence. The protein sequence
recovered shows 67% homology to the Mammalian Scratch protein available
in the database and indicative of being present on the cell surface due
to the presence of a transmembrane domain. Rest of the peptides derived
from the spectra clearly matched the sequences from Mammalian Scratch,
and therefore were pulled down as major hits. The ion fragmentation data
further confirm the identity of a novel form of Scratch as the cognate
antigen for VB3-011.

[0280] FIGS. 12 and 13 identify Mammalian Scratch as the antigen.

Discussion

[0281] VB3-011, an IgG MAb, was generated from peripheral blood
lymphocytes (PBL) isolated from a patient diagnosed with a grade II
astrocytoma, using Hybridomics® and ImmunoMine® Viventia's
proprietary platform technologies (See WO97/044461). The antibody
exhibits reactivity to a host of other cell lines each of which is
representative of different cancer indications. Despite this
demonstration of broad tumor-cell type reactivity, VB3-011 shows limited
binding to normal tissue. VB3-011 antigen was classified as a
"non-blottable" antigen with a glycan modification, attributable to CSA.

[0282] Since CSA molecules are characterized by (1-4) GlcNAc/Glucuronate
structures they also resemble the lectin-Neu5Ac (α2→6)
Gal(β1→4)Glucuronate, recognized by Hemagglutinin (HA). A new
reagent that would enable lectin-based purification was generated using.
recombinant HA was immobilized to anti-HA antibody as an purification
agent. Membrane preparations were affinity purified with immobilized-HA,
and the eluates subjected to SDS-PAGE and WB analysis, subsequently
probed with VB3-011 antibody. VB3-011 detected a ˜50 kDa protein on
1D-PAGE that further resolved into a ˜36 kDa band on 2D-PAGE
analysis. LC-MS/MS analysis of the 1D and 2D spots identified Mammalian
Scratch as the antigen with molecular weight 36 kDa (of ˜50 kDa
observed by WB analysis of 1D-PAGE), thus attributing the rest to the
presence of the glycan, 4-sulfated, Neu5Ac (α2→6)
Gal(β1→4)Glucuronate. The detection of a 36 kDa spot on
2D-PAGE matched the molecular weight and isoelectric point [(pI), i.e.,
9.7±0.2] characteristic of Mammalian Scratch.

[0283] The protein sequences recovered by denovo sequencing from MS/MS
fragment ion analyses, resulted in 67% coverage with 16 out of 17
peptides showing 100% homology to the Mammalian Scratch sequence found in
the database (gi|13775236). One peptide, RFLAAFLAAAGPFGFALGPSSV (SEQ ID
NO:2), from peptide mass 2134.985448, showed 100% homology in the
flanking sequences but not with the sequence in the middle, indicating an
identification of a novel sequence. The presence of this sequence is
responsible for the only transmembrane domain available on the protein
and places Scratch on the cell-surface as opposed to the cytosol. This is
the first report depicting Mammalian Scratch as a cell-surface tumor
antigen.

Example 2

Tumor Associated Expression of Scratch

[0284] An antibody specific for Mammalian Scratch was tested for tumor
specificity using HD formalin fixed TMA's. See Table 3 for normal tissues
and Table 4 for tumor specific membrane binding. There was no detection
of the Scratch antigen on the membrane of normal tissue. However,
strongly positive membrane staining was found on a variety of tumor
tissues.

Example 3

Localization of Scratch as Cancer Diagnostic

[0285] Aberrant localization of the scratch protein as an indicator of
cancer: Wild type Scratch protein has a limited expression pattern within
the nucleus of cells as described by Nakakura et al, 2001. However,
expression in the case of the tumor tissue types and cancer cell types
has been established on the membrane and within the cytoplasm of the
cells by the inventors. Using techniques known in the art such as flow
cytometery, immunohistochemistry, western blotting of membrane fractions
of cells aberrant expression of the Scratch protein and variants thereof
can be established on the membrane and within the cytoplasm of cancer
cells. This change in localization can be used as a diagnostic indicative
of cancer.

[0286] Membrane expression of variant Scratch proteins has been
established by both flow cytometery and western blotting of membrane
fractions from cancer cell types such as U-87Mg, A375, MDA-MB-435S,
U118-MG. This is shown in Table 1, and FIGS. 3, 4 and 15.

Example 4

Detection of Variant mRNA as Indication of Cancer

[0287] RT-PCR methodology for sensitive detection of variant mRNA of
Mammalian Scratch containing transmembrane domain: Messenger RNA will be
isolated from different types of tumor cells and first strand complement
DNA (cDNA) will be synthesized using the reverse transcriptase enzyme and
an oligo dT primer. The cDNA will then be used to test for the expression
of the wild type Scratch mRNA and possible variants and specifically the
transmembrane mutant by PCR using the following primers:

[0289] The cycling conditions for PCR were: 94° C. for 1 min.,
62° C. for 1 min., and 72° C. for 30 sec., for a total of
30 cycles followed by a final extension of 10 min. at 72° C.

[0290] Electrophoresis on a 1% agarose gel will demonstrate that the band
of interest of 159 bp is present in reactions using primer 1 and 140 bp
in reactions using primer 2 if the transmembrane mutant is present.

[0291] The sequence analysis of the wild type Mammalian Scratch revealed a
KpnI restriction site (position 118) that is not present in the variant.
Therefore, to test if the variant form is expressed in tumor cells, the
amplified PCR product will be digested with the KpnI restriction enzyme
followed electrophoresis on a 1.5% agarose gel. If the tumor cells
express the wild type Mammalian Scratch, then two fragments of 67 and 92
bp will be detected under UV lamp. In contrast, if the tumor cells
express a variant of Scratch, lacking the KpnI site then the size of the
PCR fragment will be identical to the undigested control. Using the
primers specific to the transmembrane region of the variant Mammalian
Scratch (primer #2) a PCR fragment will only be found in samples
containing the variant with the transmembrane domain, thereby identifying
the specific variant.

Example 5

Detection of Genomic DNA Sequence as an Indication of Cancer

[0292] The gene coding for the human Mammalian Scratch protein has been
located to chromosome 8 q24.3 and consists of 2 exons. The gene sequence
for the cancer associated membrane bound variant of the Scratch can be
easily determined using gene sequencing techniques known in the art such
as exon-specific PCR amplification, or direct DNA sequencing initiating
from primers to the known sequence.

[0293] Once the sequence of the mutated gene is known diagnostic tests
based on its detection can be used to evaluate patients. DNA chip arrays
can be created by attaching oligonucleotides corresponding to the sense
and anti-sense sequences of both wild type and the mutated gene Genomic
DNA can be isolated from the peripheral whole blood or from tumor tissues
The gene of interest is then amplified using PCR with primers
corresponding both to the wild type sequence and to the expected
mutations and labeled with an appropriate probe (usually fluorescent).
The DNA is then hybridized to the oligonucleotides on the chip and the
pattern of fluorescence determined with a fluorescent reader. By
comparing the pattern of fluorescence to a map of the known locations of
the oligonucleotides sequences the sequence of the patients gene with can
be established as either wild type or mutant. (Cooper et al 2004)

[0294] Arrays for common mutations in the p53 gene (Affymetrix) among
others are already commercially available and custom array services are
also available

Example 6

Variant Cancer Associated Scratch as a Target for Immunotoxins

[0295] VB6-011 is a immunoconjugate of modified bouganin conjugate with an
antibody that specifically recognizes Mammalian Scratch protein on the
tumor cell surface. Treatment of cells expressing variant Scratch
containing a transmembrane domain on the cell surface results in specific
uptake of the immunoconjugate and subsequent cell death.

Cytotoxicity of VB6-011 Proteins

[0296] The cytotoxicity of VB6-011 was measured by an MTS assay. Briefly,
antigen-positive and antigen-negative cells were seeded at 1000 cells per
well and incubated at 37° C. for 3 hours. Subsequently, varying
concentrations of VB6-011 and de-bouganin were added to the cells and
after 5 days, the cell viability determined.

[0297] The negative and positive-antigen cell lines were incubated with
different concentrations of VB6-011 from 1 nM to 1 mM. After 5 days
incubation, the calculated IC50 of VB6-011 was 350 nM. (FIG. 18)
(Table 5) In contrast, no IC50 could be determined with the antigen
negative cell lines.

[0298] While the present invention has been described with reference to
what are presently considered to be the preferred examples, it is to be
understood that the invention is not limited to the disclosed examples.
To the contrary, the invention is intended to cover various modifications
and equivalent arrangements included within the spirit and scope of the
appended claims.

[0299] All publications, patents and patent applications are herein
incorporated by reference in their entirety to the same extent as if each
individual publication, patent or patent application was specifically and
individually indicated to be incorporated by reference in its entirety.

TABLE-US-00004
TABLE 1
Table 1: Increase in median fluorescence for VB3-011 over an
isotype-matched control for each cell line used in the study.
Cell line MF*
A375 11.5
U118MG 6.1
U87MG 4.6
MDA-MB-435S 4.6
PANC-1 2.1
DAUDI 1.1